Compositions and methods of use for treating metabolic disorders

ABSTRACT

A complex comprising a GDF15 polypeptide is described. Methods of treating individuals with a metabolism disorder, such as, glucose metabolism disorder and/or a body weight disorder, and compositions associated therewith, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/927,153 filed on Oct. 29, 2015, issued as U.S. Pat. No. 9,920,118,which application claims priority benefit of U.S. provisionalapplication Ser. No. 62/073,737, filed on Oct. 31, 2014 and U.S.provisional application Ser. No. 62/244,604 filed on Oct. 21, 2015,which applications are incorporated herein by reference in theirentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

A Sequence Listing is provided herewith as a text file,“NGMB-142_SeqList.txt” created on Oct. 28, 2015 and having a size of 188KB. The contents of the text file are incorporated by reference hereinin their entirety.

FIELD OF THE INVENTION

The present invention relates to, among other things, polypeptidecomplex and compositions thereof which are useful in treating metabolismrelated conditions.

INTRODUCTION

Obesity is most commonly caused by excessive food intake coupled withlimited energy expenditure and/or lack of physical exercise. Obesityincreases the likelihood of development of various diseases, such asdiabetes mellitus, hypertension, atherosclerosis, coronary arterydisease, sleep apnea, gout, rheumatism and arthritis. Moreover,mortality risk directly correlates with obesity, such that, for example,a body-mass index in excess of 40 results in an average decreased lifeexpectancy of more than 10 years.

Current pharmacological treatment modalities include appetitesuppressors targeting receptor classes (e.g., CB1, 5-HT_(2C), and NPY);regulators of the appetite circuits in the hypothalamus and themolecular actions of ghrelin; and nutrient-absorption inhibitorstargeting lipases. Unfortunately, none of the current modalities hasbeen shown to effectively treat obesity without causing adverse effects,some of which can be very severe.

High blood glucose levels stimulate the secretion of insulin bypancreatic beta-cells. Insulin in turn stimulates the entry of glucoseinto muscles and adipose cells, leading to the storage of glycogen andtriglycerides and to the synthesis of proteins. Activation of insulinreceptors on various cell types diminishes circulating glucose levels byincreasing glucose uptake and utilization, and by reducing hepaticglucose output. Disruptions within this regulatory network can result indiabetes and associated pathologic syndromes that affect a large andgrowing percentage of the human population.

Patients who have a glucose metabolism disorder can suffer fromhyperglycemia, hyperinsulinemia, and/or glucose intolerance. An exampleof a disorder that is often associated with the aberrant levels ofglucose and/or insulin is insulin resistance, in which liver, fat, andmuscle cells lose their ability to respond to normal blood insulinlevels.

In view of the prevalence and severity of obesity, diabetes andassociated metabolic and non-metabolic disorders, treatment modalitiesthat modulate, for example, appetite, glucose and/or insulin levels andenhance the biological response to fluctuating glucose levels in apatient remain of interest.

Wild type GDF15, also known as MIC-1 (macrophage inhibitory cytokine-1)has been linked to regulation of body weight (Tsai V W, et al., PLoS One2013; 8 (2): e55174; U.S. Pat. No. 8,192,735).

SUMMARY

Modified GDF15 polypeptides for treatment of metabolic disorders areprovided. The modified GDF15 polypeptides may be present in a complex. Acomplex of the present disclosure may include two GDF15 polypeptides.

In certain cases, a complex of the present invention comprises a firstpolypeptide comprising an IgG Fc sequence, the IgG Fc sequencecomprising a CH3 sequence comprising at least one engineeredprotuberance; and a second polypeptide comprising an IgG Fc sequence,the IgG Fc sequence comprising a CH3 sequence comprising at least oneengineered cavity, wherein the first polypeptide dimerizes with thesecond polypeptide via positioning of the protuberance of the firstpolypeptide into the cavity of the second polypeptide, and whereineither the C-terminus of the first polypeptide or the C-terminus of thesecond polypeptide is conjugated to the N-terminus of a GDF15 muteincomprising at least one N-linked glycosylation consensus site.

In certain cases, a complex of the present invention comprises a firstheterodimer and a second heterodimer, each of the first heterodimer andsecond heterodimer comprising a first polypeptide and a secondpolypeptide, the first polypeptide comprising an IgG Fc sequence, theIgG Fc sequence comprising a CH3 sequence comprising at least oneengineered protuberance; and the second polypeptide comprising an IgG Fcsequence, the IgG Fc sequence comprising a CH3 sequence comprising atleast one engineered cavity; wherein the first polypeptide dimerizeswith the second polypeptide via positioning of the protuberance of thefirst polypeptide into the cavity of the second polypeptide, whereineither the C-terminus the first polypeptide or the C-terminus the secondpolypeptide is conjugated to the N-terminus of a GDF15 mutein comprisingat least one N-linked glycosylation consensus site, and wherein theGDF15 mutein in the first heterodimer dimerizes with the GDF15 mutein inthe second heterodimer thereby forming the complex comprising the firstheterodimer and second heterodimer.

In exemplary embodiments, the C-terminus of the first polypeptide maybeconjugated to the N-terminus of the GDF15 mutein. In other cases, theC-terminus of the second polypeptide may be conjugated to the N-terminusof the GDF15 mutein.

Also contemplated herein is a first polypeptide comprising an IgG Fcsequence, the IgG Fc sequence comprising a CH3 sequence comprising atleast one engineered protuberance, wherein the first polypeptidedimerizes with a second polypeptide comprising an IgG Fc sequence, theIgG Fc sequence comprising a CH3 sequence comprising at least oneengineered cavity; and a GDF15 mutein comprising at least one N-linkedglycosylation consensus site, wherein the C-terminus the firstpolypeptide is conjugated to the N-terminus of the GDF15 mutein. Thefirst polypeptide may be present in a complex that may also include thesecond polypeptide.

Also disclosed herein is a first polypeptide comprising an IgG Fcsequence, the IgG Fc sequence comprising a CH3 sequence comprising atleast one engineered cavity, wherein the first polypeptide dimerizeswith a second polypeptide comprising an IgG Fc sequence, the IgG Fcsequence comprising a CH3 sequence comprising at least one engineeredprotuberance; and a GDF15 mutein comprising at least one N-linkedglycosylation consensus site, wherein the C-terminus the firstpolypeptide is conjugated to the N-terminus of the GDF15 mutein. Thefirst polypeptide may be present in a complex that may also include thesecond polypeptide.

In certain cases, the GDF15 mutein in the complex may comprise acontiguous amino acid sequence that is at least 90% identical to theamino acid sequence of wild type GDF15 (SEQ ID NO: 1). For example, theGDF15 mutein may include at least one substitution of the correspondingamino acid in SEQ ID NO: 1 that creates the N-linked glycosylationconsensus site, e.g., the substitution may be D5T or D5S. In othercases, the substitution may be R21N.

In exemplary cases, the GDF15 mutein may include at least one of thefollowing pairs of substitutions of the corresponding amino acids in SEQID NO: 1 that create the N-linked glycosylation consensus site: R16N andH18T/S; S23N and E25T/S; L24N and D26T/S; S50N and F52T/S; F52N andA54T/S; Q51N and R53T/S; R53N and A55T/S; S64N and H66T/S; L65N andR67T/S; S82N and N84T/S; K91N and D93T/S; D93N and G95T/S; T94N andV96T/S; V96N and L98T/S; S97N and Q99T/S; and A106N and D108T/S.

In exemplary cases, the GDF15 mutein may include at least one of thefollowing pairs of substitutions of the corresponding amino acids in SEQID NO: 1 that create the N-linked glycosylation consensus site: R16N andH18T; S23N and E25T; L24N and D26T; S50N and F52T; F52N and A54T; Q51Nand R53T; R53N and A55T; S64N and H66T; L65N and R67T; S82N and N84T;K91N and D93T; D93N and G95T; T94N and V96T; V96N and L98T; S97N andQ99T; and A106N and D108T.

In some cases, the GDF15 mutein may include at least one of thefollowing pairs of substitutions of the corresponding amino acids in SEQID NO: 1 that create the N-linked glycosylation consensus site: R16N andH18S; S23N and E25S; L24N and D26S; S50N and F52S; F52N and A54S; Q51Nand R53S; R53N and A55S; S64N and H66S; L65N and R67S; S82N and N84S;K91N and D93S; D93N and G95S; T94N and V96S; V96N and L98S; S97N andQ99S; and A106N and D108S.

In certain embodiments, the GDF15 mutein may include at least one of thefollowing pairs of substitutions of the corresponding amino acids in SEQID NO: 1: S23N and E25T/S; R53N and A55T/S; S64N and H66T/S; K91N andD93T/S; D93N and G95T/S; S97N and Q99T/S; and A106N and D108T/S.

In certain embodiments, the GDF15 mutein may include at least one of thefollowing pairs of substitutions of the corresponding amino acids in SEQID NO: 1: S23N and E25T; R53N and A55T; S64N and H66T; K91N and D93T;D93N and G95T; S97N and Q99T; and A106N and D108S.

In certain embodiments, the GDF15 mutein may include at least one of thefollowing pairs of substitutions of the corresponding amino acids in SEQID NO: 1: S23N and E25S; R53N and A55S; S64N and H66S; K91N and D93S;D93N and G95S; S97N and Q99S; and A106N and D108S.

In certain embodiments, the GDF15 mutein may include at least one of thefollowing pairs of substitutions of the corresponding amino acids in SEQID NO: 1: S64N and H66T/S; K91N and D93T/S; D93N and G95T/S; and S97Nand Q99T/S. For example, the GDF15 mutein may include at least one ofthe following pairs of substitutions of the corresponding amino acids inSEQ ID NO: 1: S64N and H66T; K91N and D93T; D93N and G95T; and S97N andQ99T; or S64N and H66S; K91N and D93S; D93N and G95S; and S97N and Q99S.

In other embodiments, the GDF15 mutein may include at least one of thefollowing pairs of substitutions of the corresponding amino acids in SEQID NO: 1: K91N and D93T or D93S; and D93N and G95T or G95S.

In other embodiments, the GDF15 mutein in the complex may comprise acontiguous amino acid sequence that may be at least 98 amino acids longand may be at least 90% identical to the amino acid sequence of SEQ IDNO: 1, where the C-terminal amino acid of the GDF15 mutein correspondsto Isoleucine at position 112 in SEQ ID NO: 1.

In other embodiments, the contiguous amino acid sequence may be at least98 amino acids long and may be at least 95% identical to the amino acidsequence of SEQ ID NO: 1, where the C-terminal amino acid of the GDF15mutein corresponds to Isoleucine at position 112 in SEQ NO: 1.

Exemplary GDF15 mutein present in the complex disclosed herein include acontiguous amino acid sequence that is at least 98 amino acids long, atleast 90% identical to the amino acid sequence of SEQ ID NO: 1, and havedeletions of amino acids relative to SEQ ID NO: 1. For example, thepolypeptides may have an N-terminal truncation relative to SEQ ID NO: 1.The N-terminal truncation may be of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or more amino acids relative to SEQ ID NO: 1, e.g., 1-14amino acids, 2-14 amino acids, 3-14 amino acids, 2-3 amino acids, 3-5amino acids, or 4-6 amino acids.

Exemplary complexes disclosed herein include the GDF15 mutein thatincludes a contiguous amino acid sequence at least 98 amino acids longand at least 95% identical to the amino acid sequence of SEQ ID NO: 1,wherein the C-terminal amino acid of the polypeptide corresponds toIsoleucine at position 112 in SEQ ID NO: 1.

In certain cases, the contiguous amino acid sequence in the GDF15 muteinis at least 98 amino acids long and does not include the first aminoacid that corresponds to the first amino acid present at the N-terminusof SEQ ID NO: 1, wherein the C-terminal amino acid corresponds toIsoleucine at position 112 in SEQ ID NO: 1.

In certain cases, the contiguous amino acid sequence in the GDF15 muteinis at least 98 amino acids long and does not include the first two aminoacids that correspond to the first two amino acids present at theN-terminus of SEQ ID NO: 1, wherein the C-terminal amino acidcorresponds to Isoleucine at position 112 in SEQ ID NO: 1.

In certain cases, the contiguous amino acid sequence is at least 98amino acids long and does not include the first three amino acids thatcorrespond to the first three amino acids present at the N-terminus ofSEQ ID NO: 1, wherein the C-terminal amino acid corresponds toIsoleucine at position 112 in SEQ ID NO: 1.

In certain cases, the contiguous amino acid sequence is at least 98amino acids long and does not include the first six amino acids thatcorrespond to the first six amino acids present at the N-terminus of SEQID NO: 1, wherein the C-terminal amino acid corresponds to Isoleucine atposition 112 in SEQ ID NO: 1.

In certain cases, the contiguous amino acid sequence is at least 98amino acids long and does not include the first fourteen amino acidsthat correspond to the first fourteen amino acids present at theN-terminus of SEQ ID NO: 1.

In certain cases, the C-terminus of either the first polypeptide (forexample, Fc-knob) or the second polypeptide (for example, Fc-hole) isconjugated to the N-terminus of the GDF15 mutein via a linker. Exemplarylinkers include the sequence (G₄S)_(n), wherein n=1-10, e.g., 1-5 or2-5, for examples 2, 3, 4, or 5.

In certain cases, the IgG Fc comprises a contiguous amino acid sequenceat least 90% identical to the amino acid sequence in SEQ ID NO: 2 (humanIgG1 Fc sequence). The engineered protuberance may include at least onesubstitution of the corresponding amino acid in a human IgG1 Fcsequence, wherein the substitution is at a position selected from thegroup consisting of amino acid residues 347, 366 and 394, according toEU numbering. For example, the at least one substitution is selectedfrom the group consisting of Q347W/Y, T366W/Y, and T394W/Y, according toEU numbering.

In certain cases, the engineered cavity comprises at least onesubstitution of the corresponding amino acid in a human IgG1 Fcsequence, wherein the substitution is at a position selected from thegroup consisting of amino acid residues 366, 368, 394, 405, and 407,according to EU numbering. For example, the least one substitution isselected from the group consisting of T366S, L368A, T394S, F405T/V/A,and Y407T/V/A, according to EU numbering.

In certain cases, the protuberance may include the substitution T366W/Yand the cavity may include the substitutions T366S, L368A, andY407T/V/A, according to EU numbering.

For example, the protuberance may include the substitution T366W/Y andthe cavity may include the substitution Y407T/V/A, according to EUnumbering. In other cases, the protuberance may include the substitutionT366Y and the cavity may include the substitution Y407T, according to EUnumbering. In other examples, the protuberance may include thesubstitution T366W and the cavity may include the substitution Y407A,according to EU numbering. In further examples, the protuberance mayinclude the substitution T394Y and the cavity may include thesubstitution Y407T, according to EU numbering.

In certain embodiments, the IgG Fc sequences of the first and secondpolypeptides may each include a hinge region that forms at least onedisulphide bond between the first and second polypeptides. In certainembodiments, the IgG Fc sequences of the first and second polypeptidesmay each include a hinge region, a CH2 region, and a CH3 region, wherethe hinge regions form at least one disulphide bond between the firstand second polypeptides.

Also provided herein is a nucleic acid molecule encoding the abovedescribed first and second polypeptides. The nucleic acid molecule maybe operably linked to an expression control element that confersexpression of the nucleic acid molecule encoding the polypeptides invitro or in vivo. A vector that includes the nucleic acid molecule isalso contemplated. The vector may be a viral vector. In certain cases, afirst nucleic acid encoding the first polypeptide and a second nucleicacid encoding the second polypeptide are provided. Each of the nucleicacids is operably linked to an expression control element that confersexpression of the first and second polypeptides from the first andsecond nucleic acids, respectively. A first vector comprising the firstnucleic and a second vector comprising the second nucleic acid is alsodisclosed. As noted here, the vector may be a viral vector.

Some embodiments include transformed host cells that express one or moreof the aforementioned polypeptides. For example, a host cell thatincludes the first and second nucleic acids is provided. The host cellexpresses the first polypeptide and the second polypeptide.

In particular embodiments of the present disclosure, one or more of theaforementioned complexes is formulated to yield a pharmaceuticalcomposition, wherein the composition also includes one or morepharmaceutically acceptable diluents, carriers or excipients. In certainembodiments, a pharmaceutical composition also includes at least oneadditional prophylactic or therapeutic agent.

Also provided is a composition (for example, pharmaceutical composition)of one or more of the aforementioned complexes for treating orpreventing a body weight disorder in a subject; for treating orpreventing a glucose metabolism disorder in a subject. The compositionmay include an amount of the complex that is effective for treating orpreventing a body weight disorder in a subject. The composition mayinclude an amount of the complex that is effective for treating orpreventing a glucose metabolism disorder in a subject.

Still further embodiments of the present disclosure comprise an antibodythat binds specifically to one of the aforementioned first or secondpolypeptides.

Furthermore, the present disclosure contemplates pharmaceuticalcompositions comprising an antibody as described above formulated withat least one pharmaceutically acceptable excipient, carrier or diluent.Such pharmaceutical compositions may also contain at least oneadditional prophylactic or therapeutic agent.

Certain embodiments of the present disclosure contemplate a sterilecontainer that contains one of the above-mentioned pharmaceuticalcompositions and optionally one or more additional components. By way ofexample, but not limitation, the sterile container may be a syringe. Instill further embodiments, the sterile container is one component of akit; the kit may also contain, for example, a second sterile containerthat contains at least one prophylactic or therapeutic agent.

Also disclosed herein is a method of making the aforementionedpolypeptides and complexes. The method may include culturing a host cellexpressing the polypeptides; and isolating the complex that includes theexpressed polypeptides.

The present disclosure also contemplates a method of treating orpreventing a glucose metabolism disorder in a subject (e.g., a human) byadministering to the subject a therapeutically effective amount of theaforementioned complex. In some methods, the treating or preventingresults in a reduction in plasma glucose in the subject, a reduction inplasma insulin in the subject, a reduction in body weight and/or foodintake, or an increase in glucose tolerance in the subject. Inparticular embodiments, the glucose metabolism disorder is diabetesmellitus.

A method of treating or preventing a body weight disorder in a subjectis also disclosed. The method may include administering to the subjectthe complex of the present disclosure, wherein the complex isadministered in an amount effective in treating or preventing the bodyweight disorder in the subject. In some methods, the treating orpreventing results in a reduction in body weight and/or food intake inthe subject.

In some embodiments, the subject is obese and/or has a body weightdisorder.

Though not limited to any particular route of administration or dosingregimen, in some embodiments the administering is by parenteral (e.g.,subcutaneous) injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a cartoon schematic of homodimeric complex ofheterodimers of (Fc/Fc)-GDF15 molecules where the Fc/Fc polypeptides areknob-in-hole Fc pairs (A-F) and the incorporation of N-linked glycans onthe GDF15 molecule (E, F) to enhance expression and assembly of thehomodimeric complex of the heterodimers.

FIG. 2A depicts the recoveries from Expi 293F transient expression ofengineered (Fc/Fc)-GDF15 complexes. Recoveries are as follows:(0=aggregates/no expression, <25 mg/L, 25 mg/L-49.9 mg/L, 50 mg/L-74.9mg/L, 75 mg/L-99.0 mg/L, >100 mg/L). The addition of a N-linked glycanon the GDF15 sequence in the (Fc/Fc)-GDF15 provides a significantincrease to overall recoveries following purification. FIG. 2B providesthe recoveries from Expi 293F transient expression of wild-type GDF15and GDF15-glycosylation mutants (glycomuteins) that were not conjugatedto Fc.

FIG. 3 depicts the reduction in body weight in diet-induced obese (DIO)mouse model upon subcutaneous delivery of 0.4 nmol/kg of (Fc/Fc)-GDF15complexes, once a week for 4 weeks, followed by a 14 day recoveryperiod. B13a/B13b variant has significantly improved efficacy comparedto B9a/B9b and B11a/B11b variants.

FIG. 4 depicts the percent reduction in body weight in DIO mouse modelupon subcutaneous delivery of 0.4 nmol/kg of (Fc/Fc)-GDF15 complex, oncea week for 4 weeks, followed by a 14 day recovery period. B13a/B13bvariant has a vehicle subtracted % change in body weight of greater than20% after 14 days of recovery following dosing.

FIG. 5 depicts the reduction in body weight in DIO mouse model uponsubcutaneous delivery of 4.0 nmol/kg of (Fc/Fc)-GDF15 complexes, once aweek for 4 weeks, followed by a 14 day recovery period.

FIG. 6 depicts the percent reduction in body weight in DIO mouse modelupon subcutaneous delivery of 4.0 nmol/kg of (Fc/Fc)-GDF15 complexes,once a week for 4 weeks, followed by a 14 day recovery period.

FIGS. 7 and 8 summarize the observed body weight decreases (includingSEM and p-values) for each group of DIO mice (n=6) for 0.4 nmol/kg and4.0 nmol/kg dose groups depicted in FIGS. 3 and 5. For all groups,(*=p<0.05, **=p<0.01 and ***=p<0.001) via unpaired t-test.

FIG. 9 summarizes the percent body weight decreases (including SEM andp-values) for each group of DIO mice (n=6) for 0.4 nmol/kg and 4.0nmol/kg dose groups depicted in FIGS. 4 and 6. For all groups,(*=p<0.05, **=p<0.01 and ***=p<0.001) via unpaired t-test.

DETAILED DESCRIPTION

Before the methods and compositions of the present disclosure arefurther described, it is to be understood that the disclosure is notlimited to the particular embodiments set forth herein, and it is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “thecomplex” includes reference to one or more complexes, and so forth. Itis further noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology such as “solely,” “only” andthe like in connection with the recitation of claim elements, or use ofa “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified polypeptide backbones. The terms includefusion proteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusion proteins with heterologous andhomologous leader sequences, with or without N-terminus methionineresidues; immunologically tagged proteins; and the like. In specificembodiments, the terms refer to a polymeric form of amino acids of anylength which include genetically coded amino acids. In particularembodiments, the terms refer to a polymeric form of amino acids of anylength which include genetically coded amino acids fused to aheterologous amino acid sequence. In particular embodiments, the termsrefer to an amino acid of 98-112 amino acids in length, optionally fusedto a heterologous sequence. In specific embodiments, as appropriate,when referring to proteins and molecules disclosed and described herein,the terms “polypeptide,” “peptide,” and “protein” refer to Polypeptidesas defined herein.

The term “complex” as used herein refers to a protein complex thatcomprises at least two polypeptides, each of which polypeptides comprisean N-terminus and a C-terminus. The at least two polypeptides may beassociated with each other via one or both of a covalent and anon-covalent interaction (e.g., electrostatic, π-effects, van der Waalsforces, and hydrophobic effects). The at least two polypeptides may bethe same, i.e., have identical amino acid sequence or may be different,i.e., not have identical amino acid sequences. A complex having twopolypeptides, where both the polypeptides are identical, is referred toas a homodimer. A complex having two polypeptides, where thepolypeptides are different, is referred to as a heterodimer. A complexhaving three polypeptides, where the three polypeptides are identical,is referred to as a homotrimer. A complex having three polypeptides,where at least one of the three polypeptides is different from the otherpolypeptide(s), is referred to as a heterotrimer. A complex having fourpolypeptides, where the four polypeptides are identical, is referred toas a homotetramer. A complex having four polypeptides, where at leastone of the four polypeptides is different from the other polypeptide(s),is referred to as a heterotetramer. An exemplary complex of fourpolypeptides-two molecules of a first polypeptide and two molecules of asecond polypeptide, where the first polypeptide dimerizes with thesecond polypeptide to form a heterodimer and where two such heterodimersdimerize to form the complex may be referred to as a homodimeric complexof the two heterodimers.

The present disclosure contemplates complexes as defined above,including but not limited to a heterodimer having a first polypeptideassociated with a second polypeptide, where the first polypeptide is a‘knob’ Fc and the second polypeptide is a ‘hole’ Fc, and where eitherthe first polypeptide or the second polypeptide is fused to a GDF15 (orGDF15 mutein, such as, a GDF15 mutein described herein) amino acidsequence. The first and second polypeptides may be physically associatedwith each other via a non-covalent interaction (e.g., hydrophobiceffects, such as, hydrophobic interaction between the knob and holeregions of the Fc), a covalent bond (e.g., a disulfide bond, such as,one or two or more disulphide bonds between hinge regions of the Fc inthe first and second polypeptides), or both.

The present disclosure also contemplates a complex that includes twoheterodimers associated with each other, each heterodimer having a firstpolypeptide and a second polypeptide, where the first polypeptide is a‘knob’ Fc and the second polypeptide is a ‘hole’ Fc, and where eitherthe first polypeptide or the second polypeptide is fused to a GDF15 (orGDF15 mutein) amino acid sequence. Within the complex, the twoheterodimers may be physically associated by a non-covalent interaction(e.g., hydrophobic effects), a covalent bond (e.g., a disulfide bond),or both. The first and second polypeptides in each of the heterodimersin the complex may be physically associated with each other by anon-covalent interaction (e.g., hydrophobic effects), a covalent bond(e.g., a disulfide bond), or both.

The present disclosure also contemplates a complex that includes twoheterodimers associated with each other, each heterodimer having a firstpolypeptide and a second polypeptide, where the first polypeptide is a‘knob’ Fc and the second polypeptide is a ‘hole’ Fc, and where eitherthe first polypeptide or the second polypeptide is fused to a GDF15 (orGDF15 mutein) amino acid sequence. Within the complex, the twoheterodimers may be physically associated by a non-covalent interaction(e.g., hydrophobic effects) or a covalent interaction (e.g., disulfidebond(s)) between the GDF15 polypeptides and the first and secondpolypeptides in each of the heterodimers may be physically associatedwith each other by a non-covalent interaction (e.g., knob into hole), acovalent bond (e.g., a disulfide bond), or both.

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

The terms “treat”, “treating”, “treatment” and the like refer to acourse of action (such as administering a an agent, e.g., a polypeptide,a complex, or a pharmaceutical composition comprising a polypeptide, acomplex) initiated after a disease, disorder or condition, or a symptomthereof, has been diagnosed, observed, and the like so as to eliminate,reduce, suppress, mitigate, or ameliorate, either temporarily orpermanently, at least one of the underlying causes of a disease,disorder, or condition afflicting a subject, or at least one of thesymptoms associated with a disease, disorder, condition afflicting asubject. Thus, treatment includes inhibiting (i.e., arresting thedevelopment or further development of the disease, disorder or conditionor clinical symptoms association therewith) an active disease (e.g., soas to decrease the level of insulin and/or glucose in the bloodstream,to increase glucose tolerance so as to minimize fluctuation of glucoselevels, and/or so as to protect against diseases caused by disruption ofglucose homeostasis, decrease body weight, arrest increase in bodyweight).

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other caregiver that a subject requires or willbenefit from treatment. This judgment is made based on a variety offactors that are in the realm of the physician's or caregiver'sexpertise.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering an agent, e.g., a polypeptide, acomplex, or a pharmaceutical composition comprising a polypeptide, acomplex) initiated in a manner (e.g., prior to the onset of a disease,disorder, condition or symptom thereof) so as to prevent, suppress,inhibit or reduce, either temporarily or permanently, a subject's riskof developing a disease, disorder, condition or the like (as determinedby, for example, the absence of clinical symptoms) or delaying the onsetthereof, generally in the context of a subject predisposed to having aparticular disease, disorder or condition. In certain instances, theterms also refer to slowing the progression of the disease, disorder orcondition or inhibiting progression thereof to a harmful or otherwiseundesired state.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other caregiver that a subject requires or willbenefit from preventative care. This judgment is made based on a varietyof factors that are in the realm of a physician's or caregiver'sexpertise.

The phrase “therapeutically effective amount” refers to theadministration of an agent to a subject, either alone or as a part of apharmaceutical composition and either in a single dose or as part of aseries of doses, in an amount that is capable of having any detectable,positive effect on any symptom, aspect, or characteristics of a disease,disorder or condition when administered to a patient. Thetherapeutically effective amount can be ascertained by measuringrelevant physiological effects. For example, in the case of ahyperglycemic condition, a lowering or reduction of blood glucose or animprovement in glucose tolerance test can be used to determine whetherthe amount of an agent is effective to treat the hyperglycemiccondition. For example, a therapeutically effective amount is an amountsufficient to reduce or decrease any level (e.g., a baseline level) offasting plasma glucose (FPG), wherein, for example, the amount issufficient to reduce a FPG level greater than 200 mg/dl to less than 200mg/dl, wherein the amount is sufficient to reduce a FPG level between175 mg/dl and 200 mg/dl to less than the starting level, wherein theamount is sufficient to reduce a FPG level between 150 mg/dl and 175mg/dl to less than the starting level, wherein the amount is sufficientto reduce a FPG level between 125 mg/dl and 150 mg/dl to less than thestarting level, and so on (e.g., reducing FPG levels to less than 125mg/dl, to less than 120 mg/dl, to less than 115 mg/dl, to less than 110mg/dl, etc.). In the case of HbA1c levels, the effective amount is anamount sufficient to reduce or decrease levels by more than about 10% to9%, by more than about 9% to 8%, by more than about 8% to 7%, by morethan about 7% to 6%, by more than about 6% to 5%, and so on. Moreparticularly, a reduction or decrease of HbA1c levels by about 0.1%,0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%,10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more is contemplated by thepresent disclosure. The therapeutically effective amount can be adjustedin connection with the dosing regimen and diagnostic analysis of thesubject's condition and the like.

The phrase “in a sufficient amount to effect a change” means that thereis a detectable difference between a level of an indicator measuredbefore (e.g., a baseline level) and after administration of a particulartherapy. Indicators include any objective parameter (e.g., level ofglucose or insulin or food intake or body weight) or subjectiveparameter (e.g., a subject's feeling of well-being or appetite).

The phrase “glucose tolerance”, as used herein, refers to the ability ofa subject to control the level of plasma glucose and/or plasma insulinwhen glucose intake fluctuates. For example, glucose toleranceencompasses the subject's ability to reduce, within about 120 minutes,the level of plasma glucose back to a level determined before the intakeof glucose.

The terms “diabetes” and “diabetic” refer to a progressive disease ofcarbohydrate metabolism involving inadequate production or utilizationof insulin, frequently characterized by hyperglycemia and glycosuria.The terms “pre-diabetes” and “pre-diabetic” refer to a state wherein asubject does not have the characteristics, symptoms and the liketypically observed in diabetes, but does have characteristics, symptomsand the like that, if left untreated, may progress to diabetes. Thepresence of these conditions may be determined using, for example,either the fasting plasma glucose (FPG) test or the oral glucosetolerance test (OGTT). Both usually require a subject to fast for atleast 8 hours prior to initiating the test. In the FPG test, a subject'sblood glucose is measured after the conclusion of the fasting;generally, the subject fasts overnight and the blood glucose is measuredin the morning before the subject eats. A healthy subject wouldgenerally have a FPG concentration between about 90 and about 100 mg/dl,a subject with “pre-diabetes” would generally have a FPG concentrationbetween about 100 and about 125 mg/dl, and a subject with “diabetes”would generally have a FPG level above about 126 mg/dl. In the OGTT, asubject's blood glucose is measured after fasting and again two hoursafter drinking a glucose-rich beverage. Two hours after consumption ofthe glucose-rich beverage, a healthy subject generally has a bloodglucose concentration below about 140 mg/dl, a pre-diabetic subjectgenerally has a blood glucose concentration about 140 to about 199mg/dl, and a diabetic subject generally has a blood glucoseconcentration about 200 mg/dl or above. While the aforementionedglycemic values pertain to human subjects, normoglycemia, moderatehyperglycemia and overt hyperglycemia are scaled differently in murinesubjects. A healthy murine subject after a four-hour fast wouldgenerally have a FPG concentration between about 100 and about 150mg/dl, a murine subject with “pre-diabetes” would generally have a FPGconcentration between about 175 and about 250 mg/dl and a murine subjectwith “diabetes” would generally have a FPG concentration above about 250mg/dl.

The term “insulin resistance” as used herein refers to a condition wherea normal amount of insulin is unable to produce a normal physiologicalor molecular response. In some cases, a hyper-physiological amount ofinsulin, either endogenously produced or exogenously administered, isable to overcome the insulin resistance, in whole or in part, andproduce a biologic response.

The term “metabolic syndrome” refers to an associated cluster of traitsthat includes, but is not limited to, hyperinsulinemia, abnormal glucosetolerance, obesity, redistribution of fat to the abdominal or upper bodycompartment, hypertension, dysfibrinolysis, and dyslipidemiacharacterized by high triglycerides, low high density lipoprotein(HDL)-cholesterol, and high small dense low density lipoprotein (LDL)particles. Subjects having metabolic syndrome are at risk fordevelopment of Type 2 diabetes and/or other disorders (e.g.,atherosclerosis).

The phrase “glucose metabolism disorder” encompasses any disordercharacterized by a clinical symptom or a combination of clinicalsymptoms that is associated with an elevated level of glucose and/or anelevated level of insulin in a subject relative to a healthy individual.Elevated levels of glucose and/or insulin may be manifested in thefollowing diseases, disorders and conditions: hyperglycemia, type IIdiabetes, gestational diabetes, type I diabetes, insulin resistance,impaired glucose tolerance, hyperinsulinemia, impaired glucosemetabolism, pre-diabetes, other metabolic disorders (such as metabolicsyndrome, which is also referred to as syndrome X), and obesity, amongothers. The complexes of the present disclosure, and compositionsthereof, can be used, for example, to achieve and/or maintain glucosehomeostasis, e.g., to reduce glucose level in the bloodstream and/or toreduce insulin level to a range found in a healthy subject.

The term “hyperglycemia”, as used herein, refers to a condition in whichan elevated amount of glucose circulates in the blood plasma of asubject relative to a healthy individual. Hyperglycemia can be diagnosedusing methods known in the art, including measurement of fasting bloodglucose levels as described herein.

The term “hyperinsulinemia”, as used herein, refers to a condition inwhich there are elevated levels of circulating insulin when,concomitantly, blood glucose levels are either elevated or normal.Hyperinsulinemia can be caused by insulin resistance which is associatedwith dyslipidemia, such as high triglycerides, high cholesterol, highlow-density lipoprotein (LDL) and low high-density lipoprotein (HDL);high uric acids levels; polycystic ovary syndrome; type II diabetes andobesity. Hyperinsulinemia can be diagnosed as having a plasma insulinlevel higher than about 2 μU/mL.

As used herein, the phrase “body weight disorder” refers to conditionsassociated with excessive body weight and/or enhanced appetite. Variousparameters are used to determine whether a subject is overweightcompared to a reference healthy individual, including the subject's age,height, sex and health status. For example, a subject may be consideredoverweight or obese by assessment of the subject's Body Mass Index(BMI), which is calculated by dividing a subject's weight in kilogramsby the subject's height in meters squared. An adult having a BMI in therange of ˜18.5 to ˜24.9 kg/m² is considered to have a normal weight; anadult having a BMI between ˜25 and ˜29.9 kg/m² may be consideredoverweight (pre-obese); and an adult having a BMI of ˜30 kg/m² or highermay be considered obese. Enhanced appetite frequently contributes toexcessive body weight. There are several conditions associated withenhanced appetite, including, for example, night eating syndrome, whichis characterized by morning anorexia and evening polyphagia oftenassociated with insomnia, but which may be related to injury to thehypothalamus.

The term “Activators” refers to agents that, for example, stimulate,increase, activate, facilitate, enhance activation, sensitize orup-regulate the function or activity of one or more agents, e.g.,polypeptides or complex used to treat or prevent a metabolic disorder.In addition, Activators include agents that operate through the samemechanism of action as the polypeptides of the present invention (i.e.,agents that modulate the same signaling pathway as the polypeptides in amanner analogous to that of the polypeptides) and are capable ofeliciting a biological response comparable to (or greater than) that ofthe polypeptides. Examples of Activators include agonists such as smallmolecule compounds.

The term “Modulators” collectively refers to the polypeptides of thepresent invention and the Activators.

The terms “modulate”, “modulation” and the like refer to the ability ofan agent (e.g., an Activator) to increase the function or activity ofone or more polypeptides (or the nucleic acid molecules encoding them),either directly or indirectly; or to the ability of an agent to producean effect comparable to that of one or more polypeptides.

It will be appreciated that throughout this disclosure reference is madeto amino acids according to the single letter or three letter codes. Forthe reader's convenience, the single and three letter amino acid codesare provided below:

G Glycine Gly P Proline Pro A Alanine Ala V Valine Val L Leucine Leu IIsoleucine Ile M Methionine Met C Cysteine Cys F Phenylalanine Phe YTyrosine Tyr W Tryptophan Trp H Histidine His K Lysine Lys R ArginineArg Q Glutamine Gln N Asparagine Asn E Glutamic Acid Glu D Aspartic AcidAsp S Serine Ser T Threonine Thr

As used herein, the term “variant” encompasses naturally-occurringvariants (e.g., homologs and allelic variants) andnon-naturally-occurring variants (e.g., recombinantly modified).Naturally-occurring variants include homologs, i.e., nucleic acids andpolypeptides that differ in nucleotide or amino acid sequence,respectively, from one species to another. Naturally-occurring variantsinclude allelic variants, i.e., nucleic acids and polypeptides thatdiffer in nucleotide or amino acid sequence, respectively, from oneindividual to another within a species. Non-naturally-occurring variantsinclude nucleic acids and polypeptides that comprise a change innucleotide or amino acid sequence, respectively, where the change insequence is artificially introduced, e.g., the change is generated inthe laboratory or other facility by human intervention (“hand of man”).

The term “native” or “wild type”, in reference to GDF15, refers tobiologically active, naturally-occurring GDF15, including biologicallyactive, naturally-occurring GDF15 variants. The term includes the 112amino acid human GDF15 mature sequence (SEQ ID NO: 1).

The term “muteins” as used herein refers broadly to recombinantproteins, i.e., a polypeptide comprising an artificially introducedchange in amino acid sequence, e.g., a change in amino acid sequencegenerated in the laboratory or other facility by human intervention(“hand of man”). These polypeptides usually carry single or multipleamino acid substitutions or deletions and are frequently derived fromcloned genes that have been subjected to site-directed or randommutagenesis, or from completely synthetic genes. “GDF15 Muteins” of thepresent disclosure thus encompass, for example, amino acid substitutionsand/or amino acid deletions (e.g., N-terminal truncations of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more amino acids) relative to areference polypeptide, e.g., relative to native/wild type mature humanGDF15 (SEQ ID NO: 1).

As used herein in reference to native human GDF15 or a GDF15 mutein, theterms “modified”, “modification” and the like refer to one or morechanges that modify a property of a human GDF15, a naturally-occurringGDF15 variant, or a GDF15 mutein, where the change does not alter theprimary amino acid sequence of the GDF15 polypeptide (native or mutein)itself. Such a property includes, for example, solubility, circulationhalf-life, stability, clearance, immunogenicity or allergenicity, andmanufacturability (e.g., cost and efficiency). “Modification” includes acovalent chemical modification that does not alter the primary aminoacid sequence of the GDF15 polypeptide (native or mutein) itself.Changes to human GDF15, a naturally-occurring GDF15 variant, or a GDF15mutein that may be carried out include, but are not limited to, one ormore of pegylation (covalent attachment of one or more molecules ofpolyethylene glycol (PEG), or derivatives thereof); glycosylation (e.g.,N-glycosylation), polysialylation and hesylation; maltose bindingprotein fusion; albumin fusion (e.g., HSA fusion); albumin bindingthrough, for example, a conjugated fatty acid chain (acylation);Fc-fusion; and fusion with a PEG mimetic. Some particular embodimentsentail modifications involving fusion to a Fc, and still otherparticular modifications entail modifications involving glycosylation,or a combination thereof.

The terms “DNA”, “nucleic acid”, “nucleic acid molecule”,“polynucleotide” and the like are used interchangeably herein to referto a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), complementary DNA (cDNA),recombinant polynucleotides, vectors, probes, primers and the like.

The term “probe” refers to a fragment of DNA or RNA corresponding to agene or sequence of interest, wherein the fragment has been labeledradioactively (e.g., by incorporating ³²P or ³⁵S) or with some otherdetectable molecule, such as biotin, digoxygenin or fluorescein. Asstretches of DNA or RNA with complementary sequences will hybridize, aprobe can be used, for example, to label viral plaques, bacterialcolonies or bands on a gel that contain the gene of interest. A probecan be cloned DNA or it can be a synthetic DNA strand; the latter can beused to obtain a cDNA or genomic clone from an isolated protein by, forexample, microsequencing a portion of the protein, deducing the nucleicacid sequence encoding the protein, synthesizing an oligonucleotidecarrying that sequence, radiolabeling the sequence and using it as aprobe to screen a cDNA library or a genomic library.

The term “heterologous” refers to two components that are defined bystructures derived from different sources. For example, in the contextof a polypeptide, a “heterologous” polypeptide may include operablylinked amino acid sequences that are derived from different polypeptides(e.g., a first component comprising a recombinant polypeptide and asecond component derived from a native GDF15 polypeptide). Similarly, inthe context of a polynucleotide encoding a chimeric polypeptide, a“heterologous” polynucleotide may include operably linked nucleic acidsequences that can be derived from different genes (e.g., a firstcomponent from a nucleic acid encoding a polypeptide according to anembodiment disclosed herein and a second component from a nucleic acidencoding a carrier polypeptide). Other exemplary “heterologous” nucleicacids include expression constructs in which a nucleic acid comprising acoding sequence is operably linked to a regulatory element (e.g., apromoter) that is from a genetic origin different from that of thecoding sequence (e.g., to provide for expression in a host cell ofinterest, which may be of different genetic origin than the promoter,the coding sequence or both). For example, a T7 promoter operably linkedto a polynucleotide encoding a GDF15 polypeptide or domain thereof issaid to be a heterologous nucleic acid. In the context of recombinantcells, “heterologous” can refer to the presence of a nucleic acid (orgene product, such as a polypeptide) that is of a different geneticorigin than the host cell in which it is present.

The term “operably linked” refers to linkage between molecules toprovide a desired function. For example, “operably linked” in thecontext of nucleic acids refers to a functional linkage between nucleicacid sequences. By way of example, a nucleic acid expression controlsequence (such as a promoter, signal sequence, or array of transcriptionfactor binding sites) may be operably linked to a second polynucleotide,wherein the expression control sequence affects transcription and/ortranslation of the second polynucleotide. In the context of apolypeptide, “operably linked” refers to a functional linkage betweenamino acid sequences (e.g., different domains) to provide for adescribed activity of the polypeptide.

As used herein in the context of the structure of a polypeptide,“N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxylterminus”) refer to the extreme amino and carboxyl ends of thepolypeptide, respectively, while the terms “N-terminal” and “C-terminal”refer to relative positions in the amino acid sequence of thepolypeptide toward the N-terminus and the C-terminus, respectively, andcan include the residues at the N-terminus and C-terminus, respectively.“Immediately N-terminal” or “immediately C-terminal” refers to aposition of a first amino acid residue relative to a second amino acidresidue where the first and second amino acid residues are covalentlybound to provide a contiguous amino acid sequence.

“Derived from”, in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from” aGDF15 polypeptide), is meant to indicate that the polypeptide or nucleicacid has a sequence that is based on that of a reference polypeptide ornucleic acid (e.g., a naturally occurring GDF15 polypeptide or aGDF15-encoding nucleic acid), and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made. By way ofexample, the term “derived from” includes homologues or variants ofreference amino acid or DNA sequences.

In the context of a polypeptide, the term “isolated” refers to apolypeptide of interest that, if naturally occurring, is in anenvironment different from that in which it may naturally occur.“Isolated” is meant to include polypeptides that are within samples thatare substantially enriched for the polypeptide of interest and/or inwhich the polypeptide of interest is partially or substantiallypurified. Where the polypeptide is not naturally occurring, “isolated”indicates the polypeptide has been separated from an environment inwhich it was made by either synthetic or recombinant means.

“Enriched” means that a sample is non-naturally manipulated (e.g., in alaboratory, for example, by a scientist or a clinician) so that apolypeptide of interest is present in a) a greater concentration (e.g.,at least 3-fold greater, at least 4-fold greater, at least 8-foldgreater, at least 64-fold greater, or more) than the concentration ofthe polypeptide in the starting sample, such as a biological sample(e.g., a sample in which the polypeptide naturally occurs or in which itis present after administration), or b) a concentration greater than theenvironment in which the polypeptide was made (e.g., as in a bacterialcell).

“Substantially pure” indicates that a component (e.g., a polypeptide, adimer, a tetramer, a complex) makes up greater than about 50% of thetotal content of the composition, and typically greater than about 60%of the total polypeptide content. More typically, “substantially pure”refers to compositions in which at least 75%, at least 85%, at least 90%or more of the total composition is the component of interest. In somecases, the component will make up greater than about 90%, or greaterthan about 95% of the total content of the composition.

The terms “antibodies” (Abs) and “immunoglobulins” (Igs) refer toglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules which lack antigen specificity. Antibodies are described indetail hereafter.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, that is, theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to polyclonal antibodypreparations, which can include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen.

In the context of an antibody, the term “isolated” refers to an antibodythat has been separated and/or recovered from contaminant components ofits natural environment; such contaminant components include materialswhich might interfere with diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes.

The phrase “conservative amino acid substitution” refers to substitutionof amino acid residues within the following groups: 1) L, I, M, V, F; 2)R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E.Conservative amino acid substitutions may preserve the activity of theprotein by replacing an amino acid(s) in the protein with an amino acidwith a side chain of similar acidity, basicity, charge, polarity, orsize of the side chain. Guidance for substitutions, insertions, ordeletions may be based on alignments of amino acid sequences ofdifferent variant proteins or proteins from different species.

Growth Differentiation Factor 15 (GDF15)

GDF15, also known as MIC-1 (macrophage inhibitory cytokine-1), PDF(prostate differentiation factor), PLAB (placental bone morphogeneticprotein), NAG-1 (non-steroidal anti-inflammatory drugs (NSAIDs)activated gene), TGF-PL, and PTGFB, is a member of the transforminggrowth factor β (TGF-β) super-family. GDF15, which is synthesized as a62 kDa intracellular precursor protein that is subsequently cleaved by afurin-like protease, is secreted as a 25 kDa disulfide-linked protein.(See, e.g., Fairlie et al., J. Leukoc. Biol 65:2-5 (1999)). GDF15 mRNAis seen in several tissues, including liver, kidney, pancreas, colon andplacenta, and GDF15 expression in liver can be significantlyup-regulated during injury of organs such as the liver, kidneys, heartand lungs.

The GDF15 precursor is a 308 amino acid polypeptide (NCBI Ref. Seq.NP_004855.2) containing a 29 amino acid signal peptide, a 167 amino acidpro-domain, and a mature domain of 112 amino acids which is excised fromthe pro-domain by furin-like proteases. A 308-amino acid GDF15polypeptide is referred to as a “full-length” GDF15 polypeptide; a112-amino acid GDF15 polypeptide (amino acids 197-308 of “full-length”GDF15) is a “mature” GDF15 polypeptide (SEQ ID NO: 1). Unless otherwiseindicated, the term “GDF15” refers to the 112 amino acid mature humansequence. In addition, numerical references to particular GDF15 residuesrefer to the 112 amino acid mature sequence (i.e., residue 1 is Ala (A),and residue 112 is Ile (I); see SEQ ID NO: 1). Of note, while the GDF15precursor amino acid sequence predicts three excision sites, resultingin three putative forms of “mature” human GDF15 (i.e., 110, 112 and 115amino acids), the 112 amino acid mature sequence is accepted as beingcorrect.

The scope of the present disclosure includes GDF15 orthologs, andmodified forms thereof, from other mammalian species, and their use,including mouse (NP_035949), chimpanzee (XP_524157), orangutan(XP_002828972), Rhesus monkey (EHH29815), giant panda (XP_002912774),gibbon (XP_003275874), guinea pig (XP_003465238), ferret (AER98997), cow(NP_001193227), pig (NP_001167527), dog (XP_541938) and platypus(Ornithorhynchus anatinus; AFV61279). The mature form of human GDF15 hasapproximately 67% amino acid identity to the mouse ortholog.

For the sake of convenience, the modified human GDF15 molecules, theGDF15 variants (e.g., muteins), and modified GDF15 muteins describedhenceforward are collectively referred to hereafter as the“Polypeptide(s)”. It should be noted that any reference to “human” inconnection with the Polypeptides and nucleic acid molecules of thepresent disclosure is not meant to be limiting with respect to themanner in which the Polypeptide or nucleic acid is obtained or thesource, but rather is only with reference to the sequence as it maycorrespond to a sequence of a naturally occurring human Polypeptide ornucleic acid molecule. In particular embodiments, the modified humanGDF15 molecules are N-glycosylated dimers. In addition to the humanpolypeptides and the nucleic acid molecules which encode them, thepresent disclosure contemplates GDF15-related polypeptides andcorresponding nucleic acid molecules from other species.

A. Polypeptides Having Desired Physical Properties

The present disclosure contemplates, in part, Polypeptides that includea contiguous amino acid sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQID NO: 1 (mature 112 amino acid long human GDF15). The Polypeptides mayinclude one or more amino acid substitutions and/or deletions relativeto the amino acid sequence of SEQ ID NO: 1. In certain embodiments, inaddition to the amino acid substitutions, the Polypeptides of thepresent disclosure may also include amino acid deletions relative to theamino acid sequence of SEQ ID NO: 1. In some embodiments, thePolypeptides of the present disclosure may include amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 1.

For convenience and clarity, the amino acid sequence of SEQ ID NO: 1 isused as a reference sequence for the Polypeptides presented herein.Therefore, the amino acid residue positions are numbered herein withreference to SEQ ID NO: 1. The sequence of SEQ ID NO: 1 is presentedbelow:

ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI

In some embodiments, the Polypeptides of the present disclosure mayinclude one, two, three or more amino acid substitutions, additions, ordeletions that introduce one or more N-linked glycosylation consensussite(s) at a location where such a site is not present in SEQ ID NO: 1.The N-linked glycosylation consensus site includes the sequence NXS/T,where N is Asn; X is an amino acid other than proline; followed byeither Ser (S) or Thr (T).

Examples of Polypeptides of the present disclosure include polypeptidesthat have one, two, three, four, or more glycosylation consensus sites(e.g., N-linked Glycosylation consensus sites) at an amino acid locationwhere such a site is not present in the amino acid sequence of SEQ IDNO: 1.

In certain embodiments, the polypeptide may include one amino acidsubstitution relative to SEQ ID NO: 1 that provides one N-linkedGlycosylation consensus site at the position of the substitution (e.g.,a NGD sequence in SEQ ID NO: 1 may be changed to NGT/S by onesubstitution; position of substitution underlined). In other cases, thepolypeptide may include two amino acid substitutions relative to SEQ IDNO: 1 that provide one N-linked Glycosylation consensus site at theposition of the substitutions (e.g., a KTD sequence in SEQ ID NO: 1 maybe changed to NTT/S by two substitutions; positions of substitutionsunderlined). In some embodiments, the polypeptide may include threeamino acid substitutions relative to SEQ ID NO: 1 that provide oneN-linked glycosylation consensus site at the position of thesubstitution (e.g., a GPG sequence in SEQ ID NO: 1 may be changed toNTT/S by three substitutions; position of substitutions underlined).

In certain embodiments, the polypeptide may include one or more aminoacid deletion relative to SEQ ID NO: 1 that provides an N-linkedglycosylation consensus site at the position of the deletion. Forexample, a NGDHCPLGPGRCCRLHT (SEQ ID NO: 119) sequence in SEQ ID NO: 1may be changed by deletion of amino acids D through H (underlined))thereby providing an N-linked glycosylation consensus site: NGT.

In certain embodiments, the polypeptide may include one or more aminoacid additions relative to SEQ ID NO: 1 that provides an N-linkedglycosylation consensus site at the position(s) of the addition(s). Anexample of introduction of an N-linked glycosylation consensus site byaddition of one amino acid includes adding an N to a sequence LHT in SEQID NO: 1, thereby generating the sequence LNHT, where NHT is an N-linkedglycosylation consensus site.

As noted above, the polypeptide may include one or more substitutionsrelative to SEQ ID NO: 1 and the substitutions may be numbered as theposition of the corresponding amino acid in SEQ ID NO: 1.

In certain embodiments, the Polypeptide may include a contiguous aminoacid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the amino acid sequence of SEQ ID NO: 1, where thecontiguous amino acid sequence has the substitution D5T/S or R21N.

In certain embodiments, the polypeptide may include a contiguous aminoacid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the amino acid sequence of SEQ ID NO: 1, where thecontiguous amino acid sequence has at least one of the following pairsof substitutions relative to the corresponding amino acids in SEQ ID NO:1:

i. R16N and H18T or R16N and H18S;

ii. S23N and E25T or S23N and E25S;

iii. L24N and D26T or L24N and D26S;

iv. S50N and F52T or S50N and F52S;

v. F52N and A54T or F52N and A54S;

vi. Q51N and R53T or Q51N and R53S;

vii. R53N and A55T or R53N and A55S;

viii. S64N and H66T or S64N and H66S;

ix. L65N and R67T or L65N and R67S;

x. S82N and N84T or S82N and N84S;

xi. K91N and D93T or K91N and D93S;

xii. D93N and G95T or D93N and G95S;

xiii. T94N and V96T or T94N and V96S;

xiv. V96N and L98T or V96N and L98S;

xv. S97N and Q99T or S97N and Q99S; and

xvi. A106N and D108T or A106N and D108S

For example, the substitutions in i) above, denotes that the polypeptidehas a threonine (T) or serine (S) at an amino acid position thatcorresponds to amino acid position 18 in SEQ ID NO:1, wherein in SEQ IDNO: 1 a histidine (H) is present at the amino acid position 18.Similarly, a substitution of a D at position 5 with a T or S can bedenoted by D5T/S. The position of the corresponding amino acid in apolypeptide relative to SEQ ID NO: 1 may be determined by aligning theamino acid sequences.

In certain embodiments, the polypeptide may include two amino acidsubstitutions (a pair of substitutions) that provide a singleN-glycosylation consensus sequence at a position where a N-glycosylationconsensus sequence is not present in SEQ ID NO: 1. Examples of suchsubstitutions include R16N and H18T/S; K91N and D93T/S; T94N and V96T/S;and others listed above. R16N and H18T/S denotes that the polypeptidehas a N at a position that corresponds to position 16 of SEQ ID NO: 1,where in SEQ ID NO: 1 an R is present and the polypeptide has a either Tor S at a position that corresponds to position 18 in SEQ ID NO: 1,where H is present. Since the sequence RXH (at position 16-18) in SEQ IDNO: 1 does not include any residue for the N-linked glycosylationconsensus sequence, the pair of substitutions leads to the introductionof the N-linked glycosylation consensus sequence.

In alternate embodiments, a single amino acid substitution may sufficeto provide the N-linked glycosylation consensus sequence, for example,since the sequence NGD (at position 3-5) is present in SEQ ID NO: 1, asingle substitution of D with T or S produces the sequence NGT or NGS,respectively, which are both N-glycosylation consensus sequences.

In certain cases, more than one N-glycosylation consensus sequence maybe introduced into the wild type GDF15. For example, the wild type GDF15amino acid sequence may be modified by substitutions and/or deletions toprovide one, two, three, four or more N-glycosylation consensussequences. In certain embodiments, the polypeptide may be include 112contiguous amino acids that has a sequence identity of at least 90% tothe 112 amino acids sequence of SEQ ID NO: 1, where the 112 contiguousamino acids include one, two, three, four or more N-glycosylationconsensus sequences, such as, 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2N-glycosylation consensus sequences.

In certain embodiments, the polypeptide may be include 112 contiguousamino acids that has a sequence identity of at least 90% to the 112amino acids sequence of SEQ ID NO: 1, where the 112 contiguous aminoacids include one, two, three, four or more of the pairs ofsubstitutions set forth herein.

The present disclosure also contemplates polypeptides that are activefragments (e.g., subsequences) of the polypeptides described above. Thelength of active fragments or subsequences may be 40 amino acids to 111amino acids, e.g., 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98,106, 109, or up to 111 amino acids.

The polypeptides have a defined sequence identity compared to areference sequence over a defined length of contiguous amino acids(e.g., a “comparison window”). Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology (Ausubelet al., eds. 1995 supplement)).

As an example, a suitable Polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%, amino acid sequence identity to a contiguous stretch of40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or up to 112 amino acids in SEQID NO: 1.

Exemplary fragments of the polypeptides disclosed herein includepolypeptides that have deletions of amino acids relative to SEQ IDNO: 1. For example, the polypeptides may have N-terminal truncationsand/or C-terminal truncations relative to SEQ ID NO: 1. The truncationsmay be of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more aminoacids relative to a reference polypeptide, e.g., SEQ ID NO: 1. Incertain embodiments, a polypeptide of interest may include one or moresubstitutions that introduce an N-linked glycosylation consensussequence, such as the one disclosed herein, and N-terminal truncationsand/or C-terminal truncations relative to SEQ ID NO: 1.

In certain embodiments, the polypeptide may be at least 98 amino acidslong and have an amino acid sequence identity of at least 90%, 92%, 95%,96%, 97%, 98%, 99%, or 100% to a corresponding stretch of 98 amino acidsin SEQ ID NO: 1. This polypeptide may be lacking the first two to firstfourteen amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 amino acids) present at the N-terminus of SEQ ID NO: 1, whileretaining the amino acids present at the C-terminus of SEQ ID NO:1. Inother words, the deleted amino acid(s) correspond to the N-terminusamino acids of SEQ ID NO: 1.

In certain embodiments, the GDF15 mutein may be at least 106 amino acidslong and have an amino acid sequence identity of at least 90%, 92%, 95%,96%, 97%, 98%, 99%, or 100% to a corresponding stretch of 106 aminoacids in SEQ ID NO: 1. The GDF15 mutein may be lacking the first sixamino acids present at the N-terminus of SEQ ID NO: 1.

In certain embodiments, the polypeptide may be at least 109 amino acidslong and have an amino acid sequence identity of at least 90%, 92%, 95%,96%, 97%, 98%, 99%, or 100% to a corresponding stretch of 109 aminoacids in SEQ ID NO: 1. The GDF15 mutein may be lacking the first threeamino acids present at the N-terminus of SEQ ID NO: 1.

Exemplary polypeptides of the present disclosure may include a deletionof the two N-terminal amino acids (ΔN2) relative to the WT hGDF15 andmay fused to a Fc sequence at the N-terminus. However, when referring tothe position of the amino acid substitutions, the residue numberindicated is the one that corresponds to the position in the WT maturehGDF15 (WT; SEQ ID NO: 1). Thus, the amino acid N at the N-terminus of apolypeptide missing the first two amino acids at the N-terminus may bereferred to as residue 3 although it is the first amino acid in theGDF15 mutein polypeptide amino acid sequence and preceded byheterologous amino acid sequences (e.g., Fc).

As noted above, these polypeptide fragments may include one or moreamino acid substitutions that introduce a N-glycosylation consensussequence relative to the sequence of SEQ ID NO: 1, such as, one, two, ormore of the amino acids substitutions disclosed herein.

As indicated above and as described in more detail below, thepolypeptides of the present disclosure may be modified through, forexample, pegylation (covalent attachment of one or more molecules ofpolyethylene glycol (PEG), or derivatives thereof); glycosylation (e.g.,N-glycosylation); polysialylation; albumin fusion molecules comprisingserum albumin (e.g., human serum albumin (HSA), cyno serum albumin, orbovine serum albumin (BSA)); albumin binding through, for example, aconjugated fatty acid chain (acylation); Fc-fusion; and fusion with aPEG mimetic. In certain embodiments, the modifications are introduced ina site-specific manner. In other embodiments, the modifications includea linker. The linker may conjugate the modifying moiety to thepolypeptide.

In particular embodiments, the present disclosure contemplatesmodification of mature human GDF15 and GDF15 muteins (such as thepolypeptides describe above) by conjugation with albumin. In otherembodiments, the present disclosure contemplates modification of thepolypeptides via N-glycosylation or O-glycosylation. The characteristicsof albumins and polypeptide conjugates thereof (e.g., fusion proteins),and glycosylated polypeptides are described further hereafter.

Fc-GDF15 Fusion Polypeptides and Complexes Thereof

In exemplary embodiments, the GDF15 polypeptides disclosed herein may bepresent as a fusion polypeptide comprising an Fc polypeptide or fragmentthereof fused to the amino acid sequence of one or more of thePolypeptides described herein (e.g., human GDF15 molecules, modifiedhuman GDF15 molecules, GDF15 muteins, and modified GDF15 muteins). Asprovided herein, the GDF15 polypeptide may be a wild type polypeptide ora mutein, e.g., a glycosylation mutein. As used herein, a “glycosylationmutein” or “glycomutein” or “glycosylation variant” or “glycovariant” inthe context of a polypeptide, for example, a GDF15 polypeptide refers toa polypeptide that includes one or more glycosylation consensus site ata position in the amino acid sequence at which position the reference(wild type) polypeptide does not include the glycosylation consensussite. In certain cases, the fusion polypeptide may include anFc-sequence fused to N-terminus of a GDF15 glycomutein disclosed herein.

Any Fc polypeptide sequence described herein or known in the art can bea component of the fusion proteins of the present disclosure. Thecomponents of the fusion proteins can be optionally covalently bondedthrough a linker, such as those linkers described herein. In some of theembodiments of the present disclosure, the fusion proteins comprise theFc polypeptide sequence as an N-terminal moiety and the Polypeptidesdescribed herein as a C-terminal moiety.

In certain cases, the Fc partner of the Fc-GDF15 fusion polypeptidesdisclosed herein may be an Fc having the sequence of human IgG Fc (e.g.,IgG1, IgG2, IgG3, or IgG4) or a variant thereof. Amino acid sequence ofhuman IgG1 Fc is provided as SEQ ID NO: 2:

(SEQ ID NO: 2) EPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Hinge region is italicized, CH2 domain is underlined and CH3 domain isdouble underlined. The numbering of the position of the amino acid inthe Fc sequence is according to the EU numbering (Edelman, G. M. et al.,Proc. Natl. Acad. USA, 63, 78-85 (1969)). Thus, the glutamic acidresidue “E” at position 1 in SEQ ID NO: 2 is numbered as 216; CH2 domainstarts with alanine (A) which is numbered 231; CH3 domain starts atglycine (G) which is numbered 341, according to EU numbering.

Fc partner of the Fc-GDF15 fusion polypeptides disclosed herein may bean Fc having a contiguous amino acid sequence that is at least 90%identical to SEQ ID NO: 2, for example, at least 93%, at least 95%, atleast 97%, at least 98%, or more identical to SEQ ID NO: 2. In certainembodiments, the Fc partner may be a fragment of Fc comprising a CH3domain or a contiguous amino acid sequence that is at least 90%, 92%,95%, 96%, 97%, 98%, 99%, or 100% identical to the CH3 domain in SEQ IDNO: 2. In certain embodiments, the Fc partner may be a fragment of Fccomprising a CH2 domain and a CH3 domain or a contiguous amino acidsequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the CH2 and CH3 domains in SEQ ID NO: 2. In certainembodiments, the Fc partner may be a fragment of Fc comprising a partialhinge region, a CH2 domain, and a CH3 domain or a contiguous amino acidsequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the hinge region, CH2 domain, and CH3 domain in SEQ ID NO:2. In certain embodiments, the Fc partner may have an amino acidsequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the amino acid sequence set forth in SEQ ID NO: 2.

In certain cases, the Fc partner of the Fc-GDF15 fusion polypeptides mayinclude an engineered protuberance which protuberance can associateanother Fc polypeptide that includes an engineered cavity. In othercases, the Fc partner of the Fc-GDF15 fusion polypeptides may include anengineered cavity which cavity can associate another Fc polypeptide thatincludes an engineered protuberance. Exemplary Fc sequences withengineered protuberance and/or cavity are described in U.S. Pat. No.8,216,805. In certain cases, the protuberance and the cavity may beengineered into CH3 domain of the Fc polypeptide. In certain cases, theFc partner that associates with the Fc-GDF15 fusion polypeptides of thepresent disclosure, is not conjugated to a GDF15 polypeptide.Accordingly, the Fc partner dimerizes with the Fc-GDF15 fusionpolypeptide forming a heterodimer, with one GDF15 molecule perheterodimer.

“Protuberances” or “knobs” may be engineered by replacing small aminoacid side chains in a CH3 domain of a first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory “cavities” or “holes”of identical or similar size to the protuberances are optionally createdin the CH3 domain of a second polypeptide by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine).

The “first polypeptide” may any polypeptide which is to be associatedwith a second polypeptide. The first and second polypeptide meet at an“interface” (defined below). In addition to the interface, the firstpolypeptide may comprise one or more additional domains, such as a CH2domain or a hinge region. In certain cases, the first polypeptideincludes a CH3 domain which can form the interface of the firstpolypeptide.

The “second polypeptide” may be any polypeptide which is to beassociated with the first polypeptide via an “interface”. In addition tothe interface, the second polypeptide may comprise one or moreadditional domains, such as a CH2 domain or a hinge region. In certaincases, the second polypeptide includes a CH3 domain which can form theinterface of the second polypeptide.

The “interface” comprises those “contact” amino acid residues (or othernon-amino acid groups such as carbohydrate groups, NADH, biotin, FAD orhaem group) in the first polypeptide which interact with one or more“contact” amino acid residues (or other non-amino acid groups) in theinterface of the second polypeptide. In certain cases, the interface maybe a domain of an immunoglobulin such as a constant domain (or fragmentsthereof). In certain cases, the interface comprises the CH3 domain of animmunoglobulin which is derived from an IgG antibody, for example, anhuman IgG1, IgG2, IgG3, or IgG4 antibody.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of the first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.the interface of the second polypeptide) so as to stabilize theheterodimer, and thereby favor heterodimer formation over homodimerformation, for example. The cavity may exist in the original interfaceor may be introduced synthetically (e.g. by altering nucleic acidencoding the interface). The protuberance may be introducedsynthetically (e.g. by altering nucleic acid encoding the interface) forexample, by recombinant means.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of the second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface ofthe first polypeptide. The cavity may exist in the original interface ormay be introduced synthetically (e.g. by altering nucleic acid encodingthe interface). For example, nucleic acid encoding the interface of thesecond polypeptide is altered to encode the cavity.

A protuberance is also referred to as a ‘knob’ and a cavity is alsoreferred to as a ‘hole’. Exemplary protuberances and cavities aredisclosed in U.S. Pat. No. 8,216,805 and include substitutions at thefollowing amino acid positions: 347, 366, 368, 394, 405, and 407. Thenumbering of the position of the amino acid is according to the EUnumbering. The engineered protuberance may include at least onesubstitution of the corresponding amino acid in a human IgG1 Fcsequence, wherein the substitution is at a position selected from thegroup consisting of amino acid residues 347, 366 and 394. For example,the at least one substitution is selected from the group consisting ofQ347W/Y, T366W/Y, and T394W/Y. In certain cases, the engineered cavitycomprises at least one substitution of the corresponding amino acid in ahuman IgG1 Fc sequence, wherein the substitution is at a positionselected from the group consisting of amino acid residues 366, 368, 394,405, and 407. For example, the least one substitution is selected fromthe group consisting of T366S, L368A, T394S, F405T/V/A, and Y407T/V/A.

In certain cases, the protuberance may include the substitution T366W/Yand the cavity may include the substitutions T366S, L368A, andY407T/V/A.

For example, the protuberance may include the substitution T366W/Y andthe cavity may include the substitution Y407T/V/A. In other cases, theprotuberance may include the substitution T366Y and the cavity mayinclude the substitution Y407T. In other examples, the protuberance mayinclude the substitution T366W and the cavity may include thesubstitution Y407A. In further examples, the protuberance may includethe substitution T394Y and the cavity may include the substitutionY407T.

In certain embodiments, the Fc partner of GDF15 polypeptide in thefusion polypeptide may include additional mutations that improve aproperty of the fusion polypeptide. As such, the Fc sequences in thefirst and the second polypeptides described herein, may includeadditional mutations. For example, the Fc partner sequence may include amutation(s) that abrogates (e.g., decreases or eliminates) IgG effectorfunction that otherwise may be a characteristic of the Fc partner. Incertain cases, the Fc partner sequence may include mutation(s) thatabrogate effector functions such as complement-dependent cytotoxicity(CDC), antibody-dependent cellular cytotoxicity (ADCC) andantibody-dependent cell phagocytosis (ADCP).

The four human IgG isotypes bind the activating Fcγ receptors (FcγRI,FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the firstcomponent of complement (C1q) with different affinities, yielding verydifferent effector functions. Thus, mutations within the binding regionsmay have a significant impact on effector function.

Binding of IgG to the FcγRs or C1q depends on residues located in thehinge region and the CH2 domain. Two regions of the CH2 domain arecritical for FcγRs and C1q binding, and have unique sequences in IgG2and IgG4. Substitutions into human IgG1 of IgG2 residues at positions233-236 and IgG4 residues at positions 327, 330 and 331 were shown togreatly reduce ADCC and CDC activity (Armour K L. et al., 1999. Eur JImmunol. 29(8):2613-24; Shields R L et al., 2001, J Biol Chem.276(9):6591-604). Furthermore, Idusogie et al. demonstrated that alaninesubstitution at different positions, including K322, significantlyreduced complement activation (Idusogie E E. et al., 2000. J Immunol.164(8):4178-84). Similarly, mutations in the CH2 domain of murine IgG2Awere shown to reduce the binding to FcγRI, and C1q (Steurer W. et al.,1995. J Immunol. 155(3):1165-74). In certain embodiments, the Fcpolypeptide may include a mutation in the CH2 domain that abrogates IgGeffector function(s). Exemplary mutations in the CH2 regions include:APELLGGP (SEQ ID NO: 96)

APALLGGP (SEQ ID NO: 98); APELLGGP (SEQ ID NO: 96)

APELAGGP (SEQ ID NO: 99); and APELLGGP (SEQ ID NO: 96)

APALAGGP (SEQ ID NO: 97).

In some embodiments, an Fc polypeptide conjugated to the GDF15glycomuteins comprises part or all of a wild type hinge sequence(generally at its N terminus). In some embodiments, an Fc polypeptidedoes not comprise a functional or wild type hinge sequence. In certaincases, the Fc sequence may include one of the following hinge sequences:EPKSCDKTHTCPPCP (SEQ ID NO: 100); KSCDKTHTCPPCP (SEQ ID NO: 101);SCDKTHTCPPCP (SEQ ID NO: 102); CDKTHTCPPCP (SEQ ID NO: 103); DKTHTCPPCP(SEQ ID NO: 104); KTHTCPPCP (SEQ ID NO: 105); THTCPPCP (SEQ ID NO: 106);or CPPCP (SEQ ID NO: 107); or a variant thereof having one or moresubstitutions (e.g., 1-6 substitutions, for example, 1-5, 1-4, 1, 2, 3,4, 5, or 6 substitutions). In certain cases, the Fc sequence may includehinge region that forms a covalent bond (e.g., one or more disulphidebonds) with the hinge region of another Fc. Thus, in certainembodiments, the first and second polypeptides in the complexesdisclosed herein may be associated via a covalent interaction betweenthe hinge regions of the first and second polypeptides. The covalentinteraction may include one or two intermolecular disulphide bonds.

In described in detail herein, a first polypeptide comprising an Fc knobor hole sequence conjugated to a GDF15 glycomutein is contemplated. Sucha polypeptide may be in a complex with a second Fc polypeptide withwhich the first polypeptide can physically associate via the placementof the knob into the hole of the Fc sequence.

In certain cases, a complex of a first Fc polypeptide and a second Fcpolypeptide is disclosed. One of the first or the second polypeptide maybe a fusion polypeptide of Fc and GDF15. As noted herein, the GDF15polypeptide may include a glycosylation mutation(s) leading toglycosylation of the GDF15 polypeptide. A glycosylated GDF15 polypeptidemay also be referred to as GDF15-glycan or GDF15-glycomutein. TheGDF15-glycan or GDF15-glycomutein may be as disclosed herein. In certaincases, the GDF15-glycan or GDF15-glycomutein fused to the Fc-knob orFc-hole polypeptide provided herein may be a Polypeptide that includes acontiguous amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ IDNO: 1, where the contiguous amino acid sequence has the substitutionDST; D5S; or R21N relative to the amino acid sequence of SEQ ID NO: 1.In certain embodiments, the GDF15-glycan or GDF15-glycomutein fused tothe Fc-knob or Fc-hole polypeptide may be a Polypeptide that has anamino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, wherethe amino acid sequence includes one or more of the following pairs ofsubstitutions relative to the amino acid sequence of SEQ ID NO: 1:

xvii. R16N and H18T or R16N and H185;

xviii. S23N and E25T or S23N and E25S;

xix. S50N and F52T or S50N and F52S;

xx. F52N and A54T or F52N and A54S;

xxi. R53N and A55T or R53N and A55S;

xxii. S64N and H66T or S64N and H66S;

xxiii. K91N and D93T or K91N and D93S;

xxiv. D93N and G95T or D93N and G95S;

xxv. T94N and V96T or T94N and V96S;

xxvi. V96N and L98T or V96N and L98S;

xxvii. S97N and Q99T or S97N and Q99S; and

xxviii. A106N and D108T or A106N and D108S

In certain cases, the complex may include a first and a secondpolypeptide. The first polypeptide may include an IgG Fc sequence, theIgG Fc sequence may include a CH3 sequence that includes at least oneengineered protuberance; and a second polypeptide comprising an IgG Fcsequence, the IgG Fc sequence comprising a CH3 sequence comprising atleast one engineered cavity, wherein the first polypeptide dimerizeswith the second polypeptide via positioning of the protuberance of thefirst polypeptide into the cavity of the second polypeptide, and whereineither the C-terminus of the first polypeptide or the C-terminus of thesecond polypeptide is conjugated to the N-terminus of a GDF15 muteincomprising at least one N-linked glycosylation consensus site.Accordingly, the complex comprises a heterodimer of the firstpolypeptide and the second polypeptide. Since either the first or thesecond polypeptide is fused to the GDF15 muteins disclosed herein, oneGDF15 molecule is present per heterodimer. In certain cases, the GDF15mutein may be a GDF15 mutein described herein.

As discussed herein the first and second polypeptides may interact toform a heterodimer via covalent and/or non-covalent interactions, suchas, hydrophobic interaction, disulfide bonds, or both.

In certain embodiments, a complex comprising a first heterodimer and asecond heterodimer is disclosed. Each of the first heterodimer andsecond heterodimer may include a first polypeptide and a secondpolypeptide, wherein the first polypeptide may include an IgG Fcsequence, the IgG Fc sequence may include a CH3 sequence comprising atleast one engineered protuberance; the second polypeptide may include anIgG Fc sequence, the IgG Fc sequence may include a CH3 sequencecomprising at least one engineered cavity; wherein the first polypeptidedimerizes with the second polypeptide via positioning of theprotuberance of the first polypeptide into the cavity of the secondpolypeptide, wherein either the C-terminus the first polypeptide or theC-terminus the second polypeptide is conjugated to the N-terminus of aGDF15 mutein comprising at least one N-linked glycosylation consensussite, wherein the GDF15 mutein in the first heterodimer dimerizes withthe GDF15 mutein in the second heterodimer thereby forming the complexcomprising the first heterodimer and second heterodimer. In a complex ofthe present disclosure which complex includes the first heterodimerphysically associated with a second heterodimer, two molecules of GDF15are present per heterodimer-heterodimer complex.

As noted herein, the first and second polypeptides may interact to forma heterodimer via covalent and/or non-covalent interactions, such as,hydrophobic interaction, disulfide bonds, or both and the first andsecond heterodimers dimers may interact to form a dimer-dimer complex bycovalent and/or non-covalent interactions, such as, hydrophobicinteraction, disulfide bonds, or both.

In certain cases, the GDF15 muteins present in each of the heterodimersdescribed herein, for example, in a complex of two heterodimers, may beidentical in sequence or different. In certain cases, the GDF15 muteinin a complex of two heterodimers, may be identical in sequence.

Exemplary, Fc sequences for fusion to GDF15 muteins and as bindingpartners to Fc-GDF15 fusion proteins are disclosed herein. In certainembodiments, Fc sequences present in the complexes of the presentdisclosure may be similar or identical in sequence other than theengineered ‘knob’ and ‘hole’ sequences.

In certain cases, the first and second polypeptides that may interact toform the complexes disclosed herein may be as set forth below as Pair Ithrough VIII. In the sequences set out below, the human immunoglobinG1(hIgG1) Fc sequence is followed by a linker sequence (underlined),followed by GDF15 mutein sequence (in bold).

PAIR I: First Polypeptide: hIgG1-Fc(AA)(T366W)-(G₄S)₂-ΔN2-GDF15(N3-I112) (D5T) (SEQ ID NO: 3)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KGGGGSGGGGSNGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY DDLLAKDCHCISecond Polypeptide: hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 4)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PAIR II:First Polypeptide: hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN2-GDF15 (N3-I112) (D5T)(SEQ ID NO: 5) DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS NGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCISecond Polypeptide: hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 6)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PAIR III:First Polypeptide: hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15 (G4-I112)(R21N)(SEQ ID NO: 7) DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVNASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCISecond Polypeptide: hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 8)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PAIR IV:First Polypeptide: hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15(G4-I112)(S23N/E25T) (SEQ ID NO: 9)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRANLTDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCISecond Polypeptide: hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 10)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PAIR V:First Polypeptide: hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15(G4-I112)(F52N/A54T) (SEQ ID NO: 11)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQNRTANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCISecond Polypeptide: hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 12)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PAIR VI:First Polypeptide: hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15(G4-I112)(R53N/A55T) (SEQ ID NO: 13)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFNATNMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCISecond Polypeptide: hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 14)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PAIR VII:First Polypeptide: hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15(G4-I112) (K91N/D93T) (SEQ ID NO: 15)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQNTTTGVSLQTYDDLLAKDCHCISecond Polypeptide: hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 16)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PAIR VIII:First Polypeptide: hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15(G4-I112)(D93N/G95T) (SEQ ID NO: 17)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTNTTVSLQTYDDLLAKDCHCISecond Polypeptide: hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 18)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In certain cases, the first and second polypeptides that may interact toform the complexes disclosed herein may have an amino acid sequence atleast 80% identical to the amino acid sequence of the first and secondpolypeptides, respectively, as disclosed above in Pairs I through VIII.For example, the sequence identity may be at least 85%, at least 90%, atleast 95%, at least 97%, at least 99%, or more.

In certain embodiments, the complex may include a first polypeptidehaving an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%,99%, or 100% identical to amino acid sequence of SEQ ID NO: 3; and asecond polypeptide having an amino acid sequence at least 90%, 92%, 95%,96%, 97%, 98%, 99%, or 100% identical to amino acid sequence of SEQ IDNO:4, where the first and second polypeptides are covalently linked viaat least one intermolecular disulphide bond. Also provided herein is acomplex comprising a first heterodimer and a second heterodimer, each ofthe first heterodimer and second heterodimer comprising a firstpolypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%,97%, 98%, 99%, or 100% identical to amino acid sequence of SEQ ID NO: 3;and a second polypeptide having an amino acid sequence at least 90%,92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid sequenceof SEQ ID NO:4.

In certain embodiments, the complex may include a first polypeptidehaving an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%,99%, or 100% identical to amino acid sequence of SEQ ID NO: 5; and asecond polypeptide having an amino acid sequence at least 90%, 92%, 95%,96%, 97%, 98%, 99%, or 100% identical to amino acid sequence of SEQ IDNO:6, where the first and second polypeptides are covalently linked viaat least one intermolecular disulphide bond. Also provided herein is acomplex comprising a first heterodimer and a second heterodimer, each ofthe first heterodimer and second heterodimer comprising a firstpolypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%,97%, 98%, 99%, or 100% identical to amino acid sequence of SEQ ID NO: 5;and a second polypeptide having an amino acid sequence at least 90%,92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid sequenceof SEQ ID NO:6.

In certain embodiments, the complex may include a first polypeptidehaving an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%,99%, or 100% identical to amino acid sequence of SEQ ID NO: 7; and asecond polypeptide having an amino acid sequence at least 90%, 92%, 95%,96%, 97%, 98%, 99%, or 100% identical to amino acid sequence of SEQ IDNO:8, where the first and second polypeptides are covalently linked viaat least one intermolecular disulphide bond. Also provided herein is acomplex comprising a first heterodimer and a second heterodimer, each ofthe first heterodimer and second heterodimer comprising a firstpolypeptide having an amino acid sequence at least 90%, 92%, 95%, 96%,97%, 98%, 99%, or 100% identical to amino acid sequence of SEQ ID NO: 7;and a second polypeptide having an amino acid sequence at least 90%,92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid sequenceof SEQ ID NO:8.

In particular embodiments, the complexes disclosed herein may includetwo heterodimers, each heterodimer comprising:

(a) a hIgG1-Fc polypeptide comprising a knob(Fc-knob) and having the sequence: (SEQ ID NO: 127)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; and(b) a hIgG1-Fc polypeptide comprising a hole(Fc-hole) and having the sequence: (SEQ ID NO: 4)DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,

where either the Fc-knob (a) or the Fc-hole (b) is fused at theC-terminus to the N-terminus of a GDF15 glycomutein. The sequence of theGDF15 glycomutein may be as follows:

(SEQ ID NO: 128; GDF15 (A1-I112) D5T)ARNGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI; or(SEQ ID NO: 129; ΔN2-GDF15 (N3-I112) D5T)NGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYD DLLAKDCHCI; or(SEQ ID NO: 130; ΔN3-GDF15 (G4-I112) D5T)GTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI; or(SEQ ID NO: 131; ΔN3-GDF15 (G4-I112) R21N)GDHCPLGPGRCCRLHTVNASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI; or(SEQ ID NO: 132; ΔN3-GDF15 (G4-I112) (S23N/E25T))GDHCPLGPGRCCRLHTVRANLTDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI; or(SEQ ID NO: 133; ΔN3-GDF15 (G4-I112)(F52N/A54T))GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQNRTANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI; or(SEQ ID NO: 134; ΔN3-GDF15 (G4-I112)(R53N/A55T))GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFNATNMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI; or(SEQ ID NO: 135; ΔN3-GDF15 (G4-I112) (K91N/D93T))GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQNTTTGVSLQTYDD LLAKDCHCI; or(SEQ ID NO: 136; ΔN3-GDF15 (G4-I112)(D93N/G95T))GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTNTTVSLQTYDD LLAKDCHCI.

In certain examples, the amino acid sequence of the Fc-knob may be atleast 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or more identical to aminoacid sequence of SEQ ID NO: 127. In certain examples, the amino acidsequence of the Fc-hole may be at least 85%, 90%, 92%, 95%, 96%, 97%,98%, 99%, or more identical to amino acid sequence of SEQ ID NO: 4. Incertain examples, the amino acid sequence of the GDF15 mutein may be atleast 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or more identical to aminoacid sequence of any one of SEQ ID NOs: 128-136.

The Fc-knob or the Fc-hole may be joined with the GDF15 glycomutein viaa linker sequence (G₄S)_(n), wherein n=1-10, such as, 2, 3, 4, or 5.

In certain examples, the complexes of the present disclosure may have arecovery of at least 50 mg/L, for example at least more than 55 mg/L, 60mg/L, 65 mg/L, 70 mg/L, 75 mg/L, 80 mg/L, 85 mg/L, 90 mg/L, 95 mg/L, 100mg/L, 110 mg/L, 120 mg/L, 130 mg/L, 140 mg/L, 150 mg/L, 160 mg/L, 170mg/L, 180 mg/L, 190 mg/L, 200 mg/L, or more. In certain cases, thecomplexes of the present disclosure may have a recovery of at least 50mg/L-300 mg/L, such as 60 mg/L-300 mg/L, 75 mg/L-300 mg/L, 75 mg/L-250mg/L, 75 mg/L-200 mg/L, 75 mg/L-175 mg/L, 75 mg/L-150 mg/L, 100 mg/L-300mg/L, 100 mg/L-250 mg/L, 100 mg/L-200 mg/L, 100 mg/L-150 mg/L, 100mg/L-125 mg/L, 110 mg/L-300 mg/L, or 150 mg/L-300 mg/L. Recovery of thecomplex refers to the amount of fully assembled dimer-dimer complexobtained from culture media in which a host cell expressing the firstand second polypeptides that form the two dimers present in each fullyassembled complex, is cultured.

The present disclosure also contemplates Fc polypeptide fusion partners,and fusion proteins comprising such, where the Fc polypeptide fusionpartner is modified to be one partner of a charged Fc pair. A “partnerof a charged Fc pair” refers to a (i) a “negatively charged” Fc sequence(optionally lacking the hinge region) and comprising a charged pairmutation or (ii) a “positively charged” Fc sequence (optionally lackingthe hinge region) and comprising a charged pair mutation. “Positivelycharged” and “negatively charged” are used herein for ease of referenceto describe the nature of the charge pair mutations in the Fc sequences,and not to indicate that the overall sequence or construct necessarilyhas a positive or negative charge. Charged Fc amino acid sequencessuitable for use in Polypeptide constructs (e.g., GDF15 glycomutein,modified GDF15 glycomuteins) of the present disclosure are described in,for example WO 2013/113008.

Examples of a positively charged Fc (“Fc(+)”) include an Fc comprisingan aspartatic acid-to-lysine mutation (E356K) and a glutamicacid-to-lysine mutation (D399K) of an Fc sequence lacking the hingeregion. Examples of a negatively charged Fc (“Fc(−)”) include an Fccomprising two lysine-to-aspartate mutations (K392D, K409D) in an Fcsequence lacking the hinge region. The C-terminal lysine (K477) also mayalso be optionally deleted. When a Fc(+) Polypeptide fusion protein(e.g., Fc(+) GDF15 mutein fusion protein) and a Fc(−) Polypeptide fusionprotein (e.g., Fc(−) GDF15 mutein fusion protein) are incubatedtogether, the aspartate residues associate with the lysine residuesthrough electrostatic force, facilitating formation of Fc heterodimersbetween the Fc(+) and the Fc(−) sequences of the GDF15 Polypeptidefusion proteins.

The present disclosure also contemplates constructs designated “hemi” or“hemiFc” constructs, which comprise two Fc sequences joined in tandem bya linker that connects the N-terminus of a first Fc sequence to theC-terminus of a second Fc sequence. In some embodiments, a monomercomprises a Polypeptide (e.g., a mature modified GDF15 or GDF15glycomutein) sequence linked to the first Fc sequence by a first linkerthat connects the N-terminus of the GDF15 sequence to the C-terminus ofthe first Fc sequence, wherein the first Fc sequence is linked to thesecond Fc sequence by a second linker that connects the N-terminus ofthe first Fc sequence to the C-terminus of the second Fc sequence. Thefirst and second Fc sequences also are associated by the Fc hingeregions. Two such monomers associate to form a dimer in which themonomers are linked via an interchain disulfide bond between the twoPolypeptide sequences. For examples of hemiFc polypeptides suitable foruse with the GDF15 muteins of the present disclosure see WO 2013/113008.

The present disclosure also contemplates fusion proteins having amultimer of Fc polypeptides, or fragments thereof, including a partnerof a charged Fc pair (e.g., multimer of an Fc).

The complexes of the present disclosure have improved properties such asincreased solubility, decreased aggregation, and/or increase serumhalf-life. In certain cases, the solubility of the complexes isgenerally improved relative to unconjugated recombinant human GDF15 andFc (knob or hole) conjugated wild type GDF15. In certain embodiments,the complex has a solubility of at least 1 mg/mL in phosphate bufferedsaline (PBS) at pH 7.0. In other embodiments, the complex has asolubility of at least 2 mg/mL, at least 3 mg/mL, at least 4 mg/mL, orat least 5 mg/mL. In other embodiments, the complex has a solubility ofat least 6 mg/mL in phosphate buffered saline (PBS) at pH 7.0, at least7 mg/mL, at least 8 mg/mL, at least 9 mg/mL, or at least 10 mg/mL. Inparticular embodiments, the complex has a solubility of greater than 10mg/mL.

Glycosylation: For purposes of the present disclosure, “glycosylation”is meant to broadly refer to the enzymatic process that attaches glycansto proteins, lipids or other organic molecules. The use of the term“glycosylation” in conjunction with the present disclosure is generallyintended to mean adding or deleting one or more carbohydrate moieties(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that may or may not be present in the nativesequence. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins involving a change in the natureand proportions of the various carbohydrate moieties present.

Glycosylation can dramatically affect the physical properties ofproteins and can also be important in protein stability, secretion, andsubcellular localization. Indeed, glycosylation of the GDF15 muteinpolypeptides described herein imparts beneficial improvements to theirphysical properties. By way of example, but not limitation, solubilityof GDF15 muteins can be improved by glycosylation, and such improvementmay be substantial (see Examples). The solubility improvement exhibitedby such modified GDF15 muteins can, for example, enable the generationof formulations more suitable for pharmaceutical administration thannon-glycosylated GDF15/GDF15 muteins. The glycosylated GDF15/GDF15mutein polypeptides may also exhibit enhanced stability. Moreover, thepolypeptides may improve one or more pharmacokinetic properties, such ashalf-life.

Addition of glycosylation sites can be accomplished by altering theamino acid sequence as described above. The alteration to thepolypeptide may be made, for example, by the addition of, orsubstitution by, one or more serine or threonine residues (for O-linkedglycosylation sites) or asparagine residues (for N-linked glycosylationsites). The structures of N-linked and O-linked oligosaccharides and thesugar residues found in each type may be different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (hereafterreferred to as sialic acid). Sialic acid is usually the terminal residueof both N-linked and O-linked oligosaccharides and, by virtue of itsnegative charge, may confer acidic properties to the glycoprotein. Aparticular embodiment of the present disclosure comprises the generationand use of N-glycosylation variants as described above.

Another means of increasing the number of carbohydrate moieties on thepolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide.

Dihydrofolate reductase (DHFR)—deficient Chinese Hamster Ovary (CHO)cells are a commonly used host cell for the production of recombinantglycoproteins. These cells do not express the enzyme beta-galactosidealpha-2,6-sialyltransferase and therefore do not add sialic acid in thealpha-2,6 linkage to N-linked oligosaccharides of glycoproteins producedin these cells.

In particular embodiments, the GDF15 muteins comprising at least oneN-linked glycosylation consensus site are glycosylated. Thus, inparticular embodiments, the GDF15 muteins in the complexes disclosedherein may be glycosylated at the N-linked glycosylation consensus siteintroduced into the GDF15 mutein. In certain cases, while a complexdisclosed herein may include glycosylated GDF15, such as, glycosylatedGDF15 produced during expression from a cell line, the complex may betreated post-production to remove the carbohydrate moiety. Thepost-production removal of the carbohydrate moiety may result in removalof substantially all carbohydrate groups attached to the GDF15 mutein(and the Fc sequence) during expression in a eukaryotic host cell.

Thus, the present disclosure contemplates conjugation of one or moreadditional components or molecules at the N- and/or C-terminus of apolypeptide sequence, such as another protein (e.g., a protein having anamino acid sequence heterologous to the subject protein), or a carriermolecule. Thus, an exemplary polypeptide sequence can be provided as aconjugate with another component or molecule.

A Polypeptide may also be conjugated to large, slowly metabolizedmacromolecules such as proteins; polysaccharides, such as sepharose,agarose, cellulose, cellulose beads; polymeric amino acids such aspolyglutamic acid, polylysine; amino acid copolymers; inactivated virusparticles; inactivated bacterial toxins such as toxoid from diphtheria,tetanus, cholera, leukotoxin molecules; inactivated bacteria; anddendritic cells. Such conjugated forms, if desired, can be used toproduce antibodies against a polypeptide of the present disclosure. Incertain cases, the GDF15 in the complexes described herein may bepolypeptide conjugated to a large, slowly metabolized macromolecule.

Additional candidate components and molecules for conjugation includethose suitable for isolation or purification. Particular non-limitingexamples include binding molecules, such as biotin (biotin-avidinspecific binding pair), an antibody, a receptor, a ligand, a lectin, ormolecules that comprise a solid support, including, for example, plasticor polystyrene beads, plates or beads, magnetic beads, test strips, andmembranes.

Purification methods such as cation exchange chromatography may be usedto separate conjugates by charge difference, which effectively separatesconjugates into their various molecular weights. For example, the cationexchange column can be loaded and then washed with ˜20 mM sodiumacetate, pH˜4, and then eluted with a linear (0 M to 0.5 M) NaClgradient buffered at a pH from about 3 to 5.5, e.g., at pH˜4.5. Thecontent of the fractions obtained by cation exchange chromatography maybe identified by molecular weight using conventional methods, forexample, mass spectroscopy, SDS-PAGE, or other known methods forseparating molecular entities by molecular weight.

Linkers: Any of the foregoing components and molecules used to modifythe polypeptide sequences of the present disclosure may optionally beconjugated via a linker. Suitable linkers include “flexible linkers”which are generally of sufficient length to permit some movement betweenthe modified polypeptide sequences and the linked components andmolecules. The linker molecules can be about 6-50 atoms long. The linkermolecules may also be, for example, aryl acetylene, ethylene glycololigomers containing 2-10 monomer units, diamines, diacids, amino acids,or combinations thereof. Suitable linkers can be readily selected andcan be of any suitable length, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,10-20, 20-30, 30-50 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-alanine polymers, alanine-serine polymers, glycine-serinepolymers (for example, (G_(m)S_(o))_(n), (GSGGS)_(n) (SEQ ID NO: 120),(G_(m)S_(o)G_(m))_(n), (G_(m)S_(o)G_(m)S_(o)G_(m))_(n) (SEQ ID NO: 121),(GSGGS_(m))_(n) (SEQ ID NO: 122), (GSGS_(m)G)_(n) (SEQ ID NO: 123) and(GGGS_(m))_(n) (SEQ ID NO: 124), and combinations thereof, where m, n,and o are each independently selected from an integer of at least 1 to20, e.g., 1-18, 2-16, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10), and other flexible linkers. Glycine and glycine-serine polymers arerelatively unstructured, and therefore may serve as a neutral tetherbetween components. Exemplary flexible linkers include, but are notlimited to GGSG (SEQ ID NO:21), GGSGG (SEQ ID NO:22), GSGSG (SEQ IDNO:23), GSGGG (SEQ ID NO:24), GGGSG (SEQ ID NO:25), and GSSSG (SEQ IDNO:26).

Additional flexible linkers include glycine polymers (G)_(n) orglycine-serine polymers (e.g., (GS)_(n), (GSGGS)_(n) (SEQ ID NO: 120),(GGGS)_(n) (SEQ ID NO: 125) and (GGGGS)_(n) (SEQ ID NO: 126), where n=1to 50, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50).Exemplary flexible linkers include, but are not limited to GGGS (SEQ IDNO: 19), GGGGS (SEQ ID NO: 20), GGSG (SEQ ID NO: 21), GGSGG (SEQ ID NO:22), GSGSG (SEQ ID NO: 23), GSGGG (SEQ ID NO: 24), GGGSG (SEQ ID NO:25), and GSSSG (SEQ ID NO: 26). A multimer (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 10-20, 20-30, or 30-50) of these linker sequences may belinked together to provide flexible linkers that may be used toconjugate a heterologous amino acid sequence to the Polypeptidesdisclosed herein. As described herein, the heterologous amino acidsequence may be a signal sequence and/or a fusion partner, such as,albumin, Fc sequence, and the like.

Examples of linkers include, e.g., (GGGGS)_(n) (SEQ ID NO: 126), where nis an integer from 1 to about 10 (e.g., n=1, 2, 3, 4, 5, 6, 7, 8, 9, or10); GGGSGGGSIEGR (SEQ ID NO: 48); GGGGG (SEQ ID NO: 27); EGGGS (SEQ IDNO: 28).

In some cases, the linker may be a cleavable linker, e.g., anenzymatically cleavable linker. In other cases, the linker may be anon-cleavable linker, e.g., a linker that is not cleaved enzymaticallyunder normal physiological conditions in vivo.

For example, a proteolytically cleavable linker can include a matrixmetalloproteinase (MMP) cleavage site, e.g., a cleavage site for a MMPselected from collagenase-1, -2, and -3 (MMP-1, -8, and -13), gelatinaseA and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11),matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP andMT2-MMP). Cleavage sequence of MMP-9 is Pro-X-X-Hy (wherein, Xrepresents an arbitrary residue; Hy, a hydrophobic residue) (SEQ ID NO:29), e.g., Pro-X-X-Hy-(Ser/Thr) (SEQ ID NO: 30), e.g.,Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ ID NO: 31) or Pro-Leu/Gln-Gly-Met-Thr(SEQ ID NO: 32). Another example of a protease cleavage site is aplasminogen activator cleavage site, e.g., a uPA or a tissue plasminogenactivator (tPA) cleavage site. Specific examples of cleavage sequencesof uPA and tPA include sequences comprising Val-Gly-Arg. Another exampleis a thrombin cleavage site, e.g., CGLVPAGSGP (SEQ ID NO: 33).Additional suitable linkers comprising protease cleavage sites includelinkers comprising one or more of the following amino acid sequences: 1)SLLKSRMVPNFN (SEQ ID NO: 34) or SLLIARRMPNFN (SEQ ID NO: 35), cleaved bycathepsin B; SKLVQASASGVN (SEQ ID NO: 36) or SSYLKASDAPDN (SEQ ID NO:37), cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO:38) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO: 39) cleavedby MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ ID NO: 40) cleaved by MMP-9;DVDERDVRGFASFL (SEQ ID NO: 41) cleaved by a thermolysin-like MMP;SLPLGLWAPNFN (SEQ ID NO: 42) cleaved by matrix metalloproteinase 2(MMP-2); SLLIFRSWANFN (SEQ ID NO: 43) cleaved by cathespin L;SGVVIATVIVIT (SEQ ID NO: 44) cleaved by cathepsin D; SLGPQGIWGQFN (SEQID NO: 45) cleaved by matrix metalloproteinase 1 (MMP-1); KKSPGRVVGGSV(SEQ ID NO: 46) cleaved by urokinase-type plasminogen activator;PQGLLGAPGILG (SEQ ID NO: 47) cleaved by membrane type 1matrixmetalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQID NO: 94) cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblastcollagenase and stromelysin-1; GPQGLAGQRGIV (SEQ ID NO: 49) cleaved bymatrix metalloproteinase 13 (collagenase-3); GGSGQRGRKALE (SEQ ID NO:50) cleaved by tissue-type plasminogen activator (tPA); SLSALLSSDIFN(SEQ ID NO: 51) cleaved by human prostate-specific antigen; SLPRFKIIGGFN(SEQ ID NO: 52) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ ID NO:53) cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO: 54)cleaved by calpain (calcium activated neutral protease).

In addition to the specific amino acid sequences and nucleic acidsequences provided herein, the disclosure also contemplates polypeptidesand nucleic acids having sequences that are at least 80%, at least 85%,at least 90%, or at least 95% identical in sequence to the amino acidand nucleic acids. The terms “identical” or percent “identity,” in thecontext of two or more polynucleotide sequences, or two or more aminoacid sequences, refers to two or more sequences or subsequences that arethe same or have a specified percentage of amino acid residues ornucleotides that are the same (e.g., at least 80%, at least 85%, atleast 90%, or at least 95% identical over a specified region), whencompared and aligned for maximum correspondence over a designatedregion. The disclosure specifically contemplates first and secondpolypeptide present in a complex, the first polypeptide and the secondpolypeptide having an amino acid sequence that is at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical in sequence to the amino acid sequence ofthe first and second polypeptide, respectively, of the first and secondpolypeptide pairs provided herein.

Methods of Production of Polypeptides

A polypeptide of the present disclosure can be produced by any suitablemethod, including recombinant and non-recombinant methods (e.g.,chemical synthesis).

A. Chemical Synthesis

Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS)allows the incorporation of unnatural amino acids and/or peptide/proteinbackbone modification. Various forms of SPPS, such as Fmoc and Boc, areavailable for synthesizing polypeptides of the present disclosure.Details of the chemical synthesis are known in the art (e.g., Ganesan A.2006 Mini Rev. Med. Chem. 6:3-10; and Camarero J. A. et al., 2005Protein Pept Lett. 12:723-8).

B. Recombinant Production

Where a polypeptide is produced using recombinant techniques, thepolypeptide may be produced as an intracellular protein or as a secretedprotein, using any suitable construct and any suitable host cell, whichcan be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E.coli) or a yeast host cell, respectively. Other examples of eukaryoticcells that may be used as host cells include insect cells, mammaliancells, and/or plant cells. Where mammalian host cells are used, they mayinclude human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells(e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos7 and CV1) and hamster cells (e.g., Chinese hamster ovary (CHO) cells).In specific embodiments, the Polypeptide and complexes comprising thePolypeptide is produced in CHO cells. In other embodiments, thePolypeptide and complexes comprising the Polypeptide is produced in ayeast cell and in particular embodiments may be a yeast cell geneticallyengineered to produce glycoproteins with mammalian-like N-glycans.

A variety of host-vector systems suitable for the expression of apolypeptide may be employed according to standard procedures known inthe art. See, e.g., Sambrook et al., 1989 Current Protocols in MolecularBiology Cold Spring Harbor Press, New York; and Ausubel et al. 1995Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods forintroduction of genetic material into host cells include, for example,transformation, electroporation, conjugation, calcium phosphate methodsand the like. The method for transfer can be selected so as to providefor stable expression of the introduced polypeptide-encoding nucleicacid. The polypeptide-encoding nucleic acid can be provided as aninheritable episomal element (e.g., a plasmid) or can be genomicallyintegrated. A variety of appropriate vectors for use in production of apolypeptide of interest are commercially available.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. The expressionvector provides transcriptional and translational regulatory sequences,and may provide for inducible or constitutive expression where thecoding region is operably-linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. In general, the transcriptional andtranslational regulatory sequences may include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. Promoters can be either constitutive or inducible,and can be a strong constitutive promoter (e.g., T7).

Expression constructs generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding proteins of interest. A selectablemarker operative in the expression host may be present to facilitateselection of cells containing the vector. Moreover, the expressionconstruct may include additional elements. For example, the expressionvector may have one or two replication systems, thus allowing it to bemaintained in organisms, for example, in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification. Inaddition, the expression construct may contain a selectable marker geneto allow the selection of transformed host cells. Selectable genes arewell known in the art and will vary with the host cell used.

Isolation and purification of a protein can be accomplished according tomethods known in the art. For example, a protein can be isolated from alysate of cells genetically modified to express the proteinconstitutively and/or upon induction; from culture medium in which thehost cell is grown; or from a synthetic reaction mixture, by affinitypurification, which may involve contacting the sample (cell lysate,culture medium, or reaction mixture) with an tag that specifically bindsto the protein, washing to remove non-specifically bound material, andeluting the specifically bound protein. The isolated protein can befurther purified by dialysis and other methods normally employed inprotein purification methods. In one embodiment, the protein may beisolated using metal chelate chromatography methods. Proteins maycontain modifications to facilitate isolation. In certain embodiments,the complexes of the present disclosure may be separated based on size.

In certain embodiments, a complex comprising a first polypeptide and asecond polypeptide, the first polypeptide comprising an IgG Fc sequence,the IgG Fc sequence comprising a CH3 sequence comprising at least oneengineered protuberance; the second polypeptide comprising an IgG Fcsequence, the IgG Fc sequence comprising a CH3 sequence comprising atleast one engineered cavity; where the first polypeptide dimerizes withthe second polypeptide via positioning of the protuberance of the firstpolypeptide into the cavity of the second polypeptide, where either theC-terminus the first polypeptide or the C-terminus the secondpolypeptide is conjugated to the N-terminus of a GDF15 mutein comprisingat least one N-linked glycosylation consensus site may be isolated fromthe medium in which a host cell expressing the first and secondpolypeptides is cultured.

In certain embodiments, a complex comprising a first heterodimer and asecond heterodimer, each of the first heterodimer and second heterodimercomprising a first polypeptide and a second polypeptide, the firstpolypeptide comprising an IgG Fc sequence, the IgG Fc sequencecomprising a CH3 sequence comprising at least one engineeredprotuberance; the second polypeptide comprising an IgG Fc sequence, theIgG Fc sequence comprising a CH3 sequence comprising at least oneengineered cavity; where the first polypeptide dimerizes with the secondpolypeptide via positioning of the protuberance of the first polypeptideinto the cavity of the second polypeptide, where either the C-terminusthe first polypeptide or the C-terminus the second polypeptide isconjugated to the N-terminus of a GDF15 mutein comprising at least oneN-linked glycosylation consensus site, where the GDF15 mutein in thefirst heterodimer dimerizes with the GDF15 mutein in the secondheterodimer thereby forming the complex comprising the first heterodimerand second heterodimer may be isolated from the medium in which a hostcell expressing the first and second polypeptides is cultured.

As noted herein, a first and a second nucleic acid may be present in asingle vector or separate vectors in a single host cell or two differenthost cells. In certain cases, the first and second polypeptides of thepresent disclosure may be encoded by a first and second nucleic acidrespectively that may be present expressed in the same cell. Inembodiments where the first and second nucleic acids are present indifferent cells, the cells may be fused as some point during theproduction process.

The complexes may be prepared in substantially pure or isolated form(e.g., free from other polypeptides). The complexes can be present in acomposition that is enriched for the complexes relative to othercomponents that may be present (e.g., other polypeptides or othercomplexes (e.g. homodimers, homotetramers) or other host cellcomponents). For example, purified complex (e.g., aheterodimer-heterodimer complex) may be provided such that the complexis present in a composition that is substantially free of otherexpressed proteins, e.g., less than 90%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20%, less than 10%, less than5%, or less than 1%, of the composition is made up of other expressedproteins.

Antibodies

The present disclosure provides antibodies, including isolatedantibodies that specifically bind a polypeptide or fusion protein of thepresent disclosure. The term “antibody” encompasses intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from at least two intact antibodies, andantibody binding fragments including Fab and F(ab)′₂, provided that theyexhibit the desired biological activity. The basic whole antibodystructural unit comprises a tetramer, and each tetramer is composed oftwo identical pairs of polypeptide chains, each pair having one “light”chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. In contrast, the carboxy-terminal portion of each chaindefines a constant region primarily responsible for effector function.Human light chains are classified as kappa and lambda, whereas humanheavy chains are classified as mu, delta, gamma, alpha, or epsilon, anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, and single-chainantibodies.

Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end; the constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. The antibody chains all exhibit the samegeneral structure of relatively conserved framework regions (FR) joinedby three hyper-variable regions, also called“complementarity-determining regions” or “CDRs”. The CDRs from the twochains of each pair are aligned by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4.

An intact antibody has two binding sites and, except in bifunctional orbispecific antibodies, the two binding sites are the same. A bispecificor bifunctional antibody is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments.

As set forth above, binding fragments may be produced by enzymatic orchemical cleavage of intact antibodies. Digestion of antibodies with theenzyme papain results in two identical antigen-binding fragments, alsoknown as “Fab” fragments, and an “Fc” fragment which has noantigen-binding activity. Digestion of antibodies with the enzyme pepsinresults in a F(ab′)₂ fragment in which the two arms of the antibodymolecule remain linked and comprise two-antigen binding sites. TheF(ab′)₂ fragment has the ability to crosslink antigen.

As used herein, the term “Fab” refers to a fragment of an antibody thatcomprises VH and VL regions as well as the constant domain of the lightchain and the CH1 domain of the heavy chain.

When used herein, the term “Fv” refers to the minimum fragment of anantibody that retains both antigen-recognition and antigen-bindingsites. In a two-chain Fv species, this region includes a dimer of oneheavy-chain and one light-chain variable domain in non-covalentassociation. In a single-chain Fv species, one heavy-chain and onelight-chain variable domain can be covalently linked by a flexiblepeptide linker such that the light and heavy chains can associate in a“dimeric” structure analogous to that in a two-chain Fv species. It isin this configuration that the three CDRs of each variable domaininteract to define an antigen-binding site on the surface of the VH-VLdimer. While the six CDRs, collectively, confer antigen-bindingspecificity to the antibody, even a single variable domain (or half ofan Fv comprising only three CDRs specific for an antigen) has theability to recognize and bind antigen.

When used herein, the term “complementarity determining regions” or“CDRs” refers to parts of immunological receptors that make contact witha specific ligand and determine its specificity.

The term “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion generally comprises amino acid residues from a CDR and/or thoseresidues from a “hypervariable loop”.

As used herein, the term “epitope” refers to binding sites forantibodies on protein antigens. Epitopic determinants usually comprisechemically active surface groupings of molecules such as amino acids orsugar side chains, as well as specific three-dimensional structural andcharge characteristics. An antibody is said to bind an antigen when thedissociation constant is ≤1 μM, ≤100 nM, or ≤10 nM. An increasedequilibrium constant (“K_(D)”) means that there is less affinity betweenthe epitope and the antibody, whereas a decreased equilibrium constantmeans that there is more affinity between the epitope and the antibody.An antibody with a K_(D) of “no more than” a certain amount means thatthe antibody will bind to the epitope with the given K_(D) or morestrongly. Whereas K_(D) describes the binding characteristics of anepitope and an antibody, “potency” describes the effectiveness of theantibody itself for a function of the antibody. There is not necessarilya correlation between an equilibrium constant and potency; thus, forexample, a relatively low K_(D) does not automatically mean a highpotency.

The term “selectively binds” in reference to an antibody does not meanthat the antibody only binds to a single substance, but rather that theK_(D) of the antibody to a first substance is less than the K_(D) of theantibody to a second substance. An antibody that exclusively binds to anepitope only binds to that single epitope.

When administered to humans, antibodies that contain rodent (i.e.,murine or rat) variable and/or constant regions are sometimes associatedwith, for example, rapid clearance from the body or the generation of animmune response by the body against the antibody. In order to avoid theutilization of rodent-derived antibodies, fully human antibodies can begenerated through the introduction of human antibody function into arodent so that the rodent produces fully human antibodies. Unlessspecifically identified herein, “human” and “fully human” antibodies canbe used interchangeably. The term “fully human” can be useful whendistinguishing antibodies that are only partially human from those thatare completely, or fully, human. The skilled artisan is aware of variousmethods of generating fully human antibodies.

In order to address possible human anti-mouse antibody responses,chimeric or otherwise humanized antibodies can be utilized. Chimericantibodies have a human constant region and a murine variable region,and, as such, human anti-chimeric antibody responses may be observed insome patients. Therefore, it is advantageous to provide fully humanantibodies against multimeric enzymes in order to avoid possible humananti-mouse antibody or human anti-chimeric antibody responses.

Fully human monoclonal antibodies can be prepared, for example, by thegeneration of hybridoma cell lines by techniques known to the skilledartisan. Other preparation methods involve the use of sequences encodingparticular antibodies for transformation of a suitable mammalian hostcell, such as a CHO cell. Transformation can be by any known method forintroducing polynucleotides into a host cell, including, for example,packaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) or by transfectionprocedures known in the art. Methods for introducing heterologouspolynucleotides into mammalian cells are well known in the art andinclude dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei. Mammalian cell lines available ashosts for expression are well known in the art and include, but are notlimited to, CHO cells, HeLa cells, and human hepatocellular carcinomacells.

The antibodies can be used to detect a polypeptide of the presentdisclosure. For example, the antibodies can be used as a diagnostic bydetecting the level of one or more polypeptides of the presentdisclosure in a subject, and either comparing the detected level to astandard control level or to a baseline level in a subject determinedpreviously (e.g., prior to any illness).

Therapeutic and Prophylactic Uses

The present disclosure provides methods for treating or preventingmetabolic and metabolic-associated diseases, such as, obesity and otherbody weight disorders, hyperglycemia, hyperinsulinemia, glucoseintolerance, and glucose metabolism disorders, by the administration ofthe complex of the present disclosure, or compositions thereof, asdescribed herein. Such methods may also have an advantageous effect onone or more symptoms associated with a disease, disorder or conditionby, for example, decreasing the severity or the frequency of a symptom.

In order to determine whether a subject may be a candidate for thetreatment or prevention of a body weight disorder (e.g., obesity) by themethods provided herein, parameters such as, but not limited to, theetiology and the extent of the subject's condition (e.g., how overweightthe subject is compared to reference healthy individual) should beevaluated. For example, an adult having a BMI between ˜25 and ˜29.9kg/m² may be considered overweight (pre-obese), while an adult having aBMI of ˜30 kg/m² or higher may be considered obese. As discussed herein,a complex of the present invention can effect appetite suppression, forexample, decrease appetite leading to a reduction in body weight.

In order to determine whether a subject may be a candidate for thetreatment or prevention of hyperglycemia, hyperinsulinemia, glucoseintolerance, and/or glucose disorders by the methods provided herein,various diagnostic methods known in the art may be utilized. Suchmethods include those described elsewhere herein (e.g., fasting plasmaglucose (FPG) evaluation and the oral glucose tolerance test (oGTT)).

The complexes provided herein when administered to a subject fortreating or preventing metabolic and metabolic-associated diseases, suchas, obesity and other body weight disorders, hyperglycemia,hyperinsulinemia, glucose intolerance, glucose metabolism disorders maylead to a reduction in blood glucose level, a reduction in body weight,and/or a reduction in food intake.

In certain embodiments, the complexes contemplated herein may decreaseblood glucose level, body weight, and/or food intake by at least 5%compared to that in the absence of administration of the complexes. Forexample, complexes contemplated herein may decrease blood glucose level,body weight, and/or food intake by at least 10%, 20%, 30%, 50%, 60%,70%, 80%, or 90% as compared to that prior to the start of the treatmentor prevention.

In certain embodiments, a complex of the present disclosure used totreat a metabolic disorder may be a complex that includes twoheterodimer molecules per complex, where each heterodimer is the same,and includes a first polypeptide and a second polypeptide, where thefirst polypeptide includes an IgG Fc sequence, the IgG Fc sequence mayinclude a CH3 sequence comprising at least one engineered protuberance;the second polypeptide comprising an IgG Fc sequence, the IgG Fcsequence comprising a CH3 sequence comprising at least one engineeredcavity; wherein the first polypeptide dimerizes with the secondpolypeptide via positioning of the protuberance of the first polypeptideinto the cavity of the second polypeptide to form a heterodimer, whereineither the C-terminus the first polypeptide or the C-terminus the secondpolypeptide in each heterodimer is conjugated to the N-terminus of aGDF15 mutein comprising at least one N-linked glycosylation consensussite, wherein the GDF15 mutein in the heterodimer dimerizes with theGDF15 mutein in another of the heterodimer thereby forming the complexcomprising two heterodimers.

In yet other embodiments, a complex of the present disclosure used totreat a metabolic disorder may be a complex that includes twoheterodimer molecules (heterodimer associated with heterodimer) percomplex, where each heterodimer is the same, and each heterodimerincludes a first polypeptide having an IgG Fc sequence, the IgG Fcsequence may include a CH3 sequence comprising at least one engineeredprotuberance, where the C-terminus of the firs polypeptide is fused toN-terminus of the GDF15 glycomutein; and the second polypeptidecomprising an IgG Fc sequence, the IgG Fc sequence comprising a CH3sequence comprising at least one engineered cavity; wherein the firstpolypeptide dimerizes with the second polypeptide via positioning of theprotuberance of the first polypeptide into the cavity of the secondpolypeptide to form the heterodimer, wherein the GDF15 mutein in theheterodimer dimerizes with the GDF15 mutein in another of theheterodimer thereby forming the complex comprising the two heterodimers.

Pharmaceutical Compositions

The complexes of the present disclosure may be in the form ofcompositions suitable for administration to a subject. In general, suchcompositions are “pharmaceutical compositions” comprising one or morecomplexes and one or more pharmaceutically acceptable or physiologicallyacceptable diluents, carriers or excipients. In certain embodiments, thecomplexes are present in a therapeutically effective amount in thepharmaceutical composition. The pharmaceutical compositions may be usedin the methods of the present disclosure; thus, for example, thepharmaceutical compositions can be administered ex vivo or in vivo to asubject in order to practice the therapeutic and prophylactic methodsand uses described herein. As noted herein, the complexes may or may notbe glycosylated. For example, the complexes may be glycosylated asproduced in a eukaryotic host cell and may be subject to a process forremoval of the carbohydrate moiety prior to formulation into apharmaceutical composition. The removal of carbohydrate moieties mayresult in significant reduction in glycosylation of the polypeptides inthe complexes or complete absence of glycosylation of the polypeptidesin the complexes.

In specific embodiments, the present disclosure provides methods fortreating a glucose metabolism or body weight disorder by theadministration of the complexes, N-glycosylated complexes, orcompositions thereof. In particular embodiment, the present disclosuremethods for reducing food intake or decreasing body weight by theadministration of the complexes, N-glycosylated complexes, orcompositions thereof. The present disclosure further provides a use ofthe foregoing sequences, complexes, N-glycosylated complexes, orcompositions thereof in the manufacture of a medicament for use intreating a condition selected from metabolic and metabolic-associateddiseases, such as, obesity and other body weight disorders,hyperglycemia, hyperinsulinemia, glucose intolerance, and glucosemetabolism disorders. The present disclosure further provides a use ofthe foregoing sequences, complexes, N-glycosylated complexes, orcompositions thereof in the manufacture of a medicament for use intreating a glucose metabolism or body weight disorder. The presentdisclosure further provides a use of the foregoing sequences, complexes,N-glycosylated complexes, or compositions thereof in the manufacture ofa medicament for use in reducing food intake or body weight.

Also provided herein are compositions, for example, pharmaceuticalcompositions of the sequences, complexes, and N-glycosylated complexesdisclosed herein for treating or preventing a condition selected frommetabolic and metabolic-associated diseases, such as, obesity and otherbody weight disorders, hyperglycemia, hyperinsulinemia, glucoseintolerance, and glucose metabolism disorders. The present disclosurefurther provides a composition (e.g., pharmaceutical composition) of theforegoing sequences, complexes, or N-glycosylated complexes for treatinga glucose metabolism or body weight disorder. The present disclosurefurther provides a composition (e.g., pharmaceutical composition) of theforegoing sequences, complexes, or N-glycosylated complexes for reducingfood intake or body weight.

The pharmaceutical compositions of the present disclosure can beformulated to be compatible with the intended method or route ofadministration; exemplary routes of administration are set forth herein.Furthermore, the pharmaceutical compositions may be used in combinationwith other therapeutically active agents or compounds (e.g., glucoselowering agents) as described herein in order to treat or prevent thediseases, disorders and conditions as contemplated by the presentdisclosure.

The pharmaceutical compositions typically comprise a therapeuticallyeffective amount of at least one of the complexes contemplated by thepresent disclosure and one or more pharmaceutically and physiologicallyacceptable formulation agents. Suitable pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients include, butare not limited to, antioxidants (e.g., ascorbic acid and sodiumbisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethylor n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents,dispersing agents, solvents, fillers, bulking agents, detergents,buffers, vehicles, diluents, and/or adjuvants. For example, a suitablevehicle may be physiological saline solution or citrate buffered saline,possibly supplemented with other materials common in pharmaceuticalcompositions for parenteral administration. Neutral buffered saline orsaline mixed with serum albumin are further exemplary vehicles. Thoseskilled in the art will readily recognize a variety of buffers thatcould be used in the pharmaceutical compositions and dosage forms.Typical buffers include, but are not limited to, pharmaceuticallyacceptable weak acids, weak bases, or mixtures thereof. As an example,the buffer components can be water soluble materials such as phosphoricacid, tartaric acids, lactic acid, succinic acid, citric acid, aceticacid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof.Acceptable buffering agents include, for example, a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS), andN-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

After a pharmaceutical composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready-to-use form, a lyophilized form requiring reconstitutionprior to use, a liquid form requiring dilution prior to use, or otheracceptable form. In some embodiments, the pharmaceutical composition isprovided in a single-use container (e.g., a single-use vial, ampoule,syringe, or autoinjector (similar to, e.g., an EpiPen®)), whereas amulti-use container (e.g., a multi-use vial) is provided in otherembodiments. Any drug delivery apparatus may be used to deliver thecomplexes, including implants (e.g., implantable pumps) and cathetersystems, both of which are well known to the skilled artisan. Depotinjections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the complexes disclosedherein over a defined period of time. Depot injections are usuallyeither solid- or oil-based and generally comprise at least one of theformulation components set forth herein. One of ordinary skill in theart is familiar with possible formulations and uses of depot injections.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents mentioned herein. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Acceptable diluents,solvents and dispersion media that may be employed include water,Ringer's solution, isotonic sodium chloride solution, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), and suitable mixtures thereof. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. Moreover, fatty acids such as oleic acid find use in thepreparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

The pharmaceutical compositions containing the active ingredient (e.g.,complexes of the present disclosure) may be in a form suitable for oraluse, for example, as tablets, capsules, troches, lozenges, aqueous oroily suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, or syrups, solutions, microbeads or elixirs.Pharmaceutical compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions, and such compositions may contain one ormore agents such as, for example, sweetening agents, flavoring agents,coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets, capsulesand the like contain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be, for example, diluents,such as calcium carbonate, sodium carbonate, lactose, calcium phosphateor sodium phosphate; granulating and disintegrating agents, for example,corn starch, or alginic acid; binding agents, for example starch,gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

The tablets, capsules and the like suitable for oral administration maybe uncoated or coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction. For example, a time-delay material such as glyceryl monostearateor glyceryl distearate may be employed. They may also be coated bytechniques known in the art to form osmotic therapeutic tablets forcontrolled release. Additional agents include biodegradable orbiocompatible particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides,polyglycolic acid, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers in order to control delivery of an administered composition.For example, the oral agent can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, by the useof hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems, including oil-in-water emulsions, micelles, mixed micelles, andliposomes. Methods of preparing liposomes are described in, for example,U.S. Pat. Nos. 4,235,871, 4,501,728, and 4,837,028. Methods for thepreparation of the above-mentioned formulations will be apparent tothose skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients can besuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents, for example a naturally-occurring phosphatide (e.g.,lecithin), or condensation products of an alkylene oxide with fattyacids (e.g., polyoxy-ethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols (e.g., forheptadecaethyleneoxycetanol), or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

The pharmaceutical compositions of the present disclosure may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, forexample, liquid paraffin, or mixtures of these. Suitable emulsifyingagents may be naturally-occurring gums, for example, gum acacia or gumtragacanth; naturally-occurring phosphatides, for example, soy bean,lecithin, and esters or partial esters derived from fatty acids; hexitolanhydrides, for example, sorbitan monooleate; and condensation productsof partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including implants, liposomes,hydrogels, prodrugs and microencapsulated delivery systems. For example,a time delay material such as glyceryl monostearate or glyceryl stearatealone, or in combination with a wax, may be employed.

The present disclosure contemplates the administration of the complexesin the form of suppositories for rectal administration of the drug. Thesuppositories can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include, but are not limited to,cocoa butter and polyethylene glycols.

The complexes contemplated by the present disclosure may be in the formof any other suitable pharmaceutical composition (e.g., sprays for nasalor inhalation use) currently known or developed in the future.

The concentration of a complex of polypeptides in a formulation can varywidely (e.g., from less than about 0.1%, usually at or at least about 2%to as much as 20% to 50% or more by weight) and will usually be selectedprimarily based on fluid volumes, viscosities, and subject-based factorsin accordance with, for example, the particular mode of administrationselected.

Contemplated herein is the use of Nano Precision Medical's depotdelivery technology (Nano Precision Medical; Emeryville, Calif.). Thetechnology utilizes a titania nanotube membrane that produces zero-orderrelease rates of macromolecules, such as protein and peptidetherapeutics. The biocompatible membrane is housed in a small,subcutaneous implant that provides long-term (e.g., up to one year),constant-rate delivery of therapeutic macromolecules. The technology iscurrently being evaluated for the delivery of GLP-1 agonists for thetreatment of Type II diabetes. In certain embodiments, the complex(es)disclosed herein may be a formulation with a membrane. For example, thecomplex may be impregnated into the membrane or surrounded by themembrane. The membrane may be in shape of a disc, tube or sphere. Incertain embodiments, the tube may be a nanotube or the sphere may be ananosphere.

Routes of Administration

The present disclosure contemplates the administration of the disclosedcomplexes, and compositions thereof, in any appropriate manner. Suitableroutes of administration include parenteral (e.g., intramuscular,intravenous, subcutaneous (e.g., injection or implant), intraperitoneal,intracisternal, intraarticular, intraperitoneal, intracerebral(intraparenchymal) and intracerebroventricular), oral, nasal, vaginal,sublingual, intraocular, rectal, topical (e.g., transdermal), sublingualand inhalation.

Depot injections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the complexes disclosedherein over a defined period of time. Depot injections are usuallyeither solid- or oil-based and generally comprise at least one of theformulation components set forth herein. One of ordinary skill in theart is familiar with possible formulations and uses of depot injections.

Regarding antibodies, in an exemplary embodiment an antibody or antibodyfragment of the present disclosure is stored at 10 mg/ml in sterileisotonic aqueous saline solution for injection at 4° C. and is dilutedin either 100 ml or 200 ml 0.9% sodium chloride for injection prior toadministration to the subject. The antibody is administered byintravenous infusion over the course of 1 hour at a dose of between 0.2and 10 mg/kg. In other embodiments, the antibody is administered byintravenous infusion over a period of between 15 minutes and 2 hours. Instill other embodiments, the administration procedure is viasubcutaneous bolus injection.

The present disclosure contemplates methods wherein the complexes of thepresent disclosure is administered to a subject at least twice daily, atleast once daily, at least once every 48 hours, at least once every 72hours, at least once weekly, at least once every 2 weeks, or oncemonthly.

Combination Therapy

The present disclosure contemplates the use of a complex provided hereinin combination with one or more active therapeutic agents or otherprophylactic or therapeutic modalities. In such combination therapy, thevarious active agents frequently have different mechanisms of action.Such combination therapy may be especially advantageous by allowing adose reduction of one or more of the agents, thereby reducing oreliminating the adverse effects associated with one or more of theagents; furthermore, such combination therapy may have a synergistictherapeutic or prophylactic effect on the underlying disease, disorder,or condition.

As used herein, “combination” is meant to include therapies that can beadministered separately, for example, formulated separately for separateadministration (e.g., as may be provided in a kit), and therapies thatcan be administered together in a single formulation (i.e., a“co-formulation”).

In certain embodiments, a complex is administered or appliedsequentially, e.g., where one agent is administered prior to one or moreother agents. In other embodiments, the complex is administeredsimultaneously, e.g., where two or more agents are administered at orabout the same time; the two or more agents may be present in two ormore separate formulations or combined into a single formulation (i.e.,a co-formulation). Regardless of whether the two or more agents areadministered sequentially or simultaneously, they are considered to beadministered in combination for purposes of the present disclosure.

The complexes of the present disclosure can be used in combination withother agents useful in the treatment, prevention, suppression oramelioration of the diseases, disorders or conditions set forth herein,including those that are normally administered to subjects sufferingfrom obesity, eating disorder, hyperglycemia, hyperinsulinemia, glucoseintolerance, and other glucose metabolism disorders.

The present disclosure contemplates combination therapy with numerousagents (and classes thereof), including 1) insulin, insulin mimetics andagents that entail stimulation of insulin secretion, includingsulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide,tolbutamide, glyburide, glimepiride, glipizide) and meglitinides (e.g.,repaglinide (PRANDIN) and nateglinide (STARLIX)); 2) biguanides (e.g.,metformin (GLUCOPHAGE)) and its pharmaceutically acceptable salts, inparticular, metformin hydrochloride, and extended-release formulationsthereof, such as Glumetza™, Fortamet™, and GlucophageXR™) and otheragents that act by promoting glucose utilization, reducing hepaticglucose production and/or diminishing intestinal glucose output; 3)alpha-glucosidase inhibitors (e.g., acarbose, voglibose and miglitol)and other agents that slow down carbohydrate digestion and consequentlyabsorption from the gut and reduce postprandial hyperglycemia; 4)thiazolidinediones (e.g., rosiglitazone (AVANDIA), troglitazone(REZULIN), pioglitazone (ACTOS), glipizide, balaglitazone,rivoglitazone, netoglitazone, AMG 131, MBX2044, mitoglitazone,lobeglitazone, IDR-105, troglitazone, englitazone, ciglitazone,adaglitazone, darglitazone that enhance insulin action (e.g., by insulinsensitization) including insulin, and insulin mimetics (e.g., insulindegludec, insulin glargine, insulin lispro, insulin detemir, insulinglulisine and inhalable formulations of each), thus promoting glucoseutilization in peripheral tissues; 5) glucagon-like-peptides includingDPP-IV inhibitors (e.g., alogliptin, omarigliptin, linagliptin,vildagliptin (GALVUS) and sitagliptin (JANUVIA)) and Glucagon-LikePeptide-1 (GLP-1) and GLP-1 agonists and analogs (e.g., exenatide(BYETTA and ITCA 650 (an osmotic pump inserted subcutaneously thatdelivers an exenatide analog over a 12-month period; Intarcia, Boston,Mass.)) and GLP-1 receptor agonists (e.g., dulaglutide, semaglutide,albiglutide, exenatide, liraglutide, lixisenatide, taspoglutide,CJC-1131, and BIM-51077, including intranasal, transdermal, andonce-weekly formulations thereof); 6) and DPP-IV-resistant analogues(incretin mimetics), PPAR gamma agonists, PPAR alpha agonists such asfenofibric acid derivatives (e.g., gemfibrozil, clofibrate,ciprofibrate, fenofibrate, bezafibrate), dual-acting PPAR agonists(e.g., ZYH2, ZYH1, GFT505, chiglitazar, muraglitazar, aleglitazar,sodelglitazar, and naveglitazar), pan-acting PPAR agonists, PTP1Binhibitors (e.g., ISIS-113715 and TTP814), SGLT inhibitors (e.g.,ASP1941, SGLT-3, empagliflozin, dapagliflozin, canagliflozin, BI-10773,PF-04971729, remogloflozin, TS-071, tofogliflozin, ipragliflozin, andLX-4211), insulin secretagogues, angiotensin converting enzymeinhibitors (e.g., alacepril, benazepril, captopril, ceronapril,cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril,lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril,temocapril, or trandolapril), angiotensin II receptor antagonists (e.g.,losartan i.e., COZAAR®, valsartan, candesartan, olmesartan, telmesartanand any of these drugs used in combination with hydrochlorothiazide suchas HYZAAR®) or other anti-hypertensive drugs such as LCZ 696, RXRagonists, glycogen synthase kinase-3 inhibitors, immune modulators,sympatholitics, beta-adrenergic blocking drugs (e.g., propranolol,atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartate),alpha adrenergic blocking drugs (e.g., doxazocin, prazocin or alphamethyldopa) central alpha adrenergic agonists, peripheral vasodilators(e.g. hydralazine); beta-3 adrenergic receptor agonists, llbeta-HSD1inhibitors, neutral endopeptidase inhibitors (e.g., thiorphan andphosphoramidon), aldosterone antagonists, aldosterone synthaseinhibitors, renin inhibitors (e.g. urea derivatives of di- andtri-peptides (See U.S. Pat. No. 5,116,835), amino acids and derivatives(U.S. Pat. Nos. 5,095,119 and 5,104,869), amino acid chains linked bynon-peptidic bonds (U.S. Pat. No. 5,114,937), di- and tri-peptidederivatives (U.S. Pat. No. 5,106,835), peptidyl amino diols (U.S. Pat.Nos. 5,063,208 and 4,845,079) and peptidyl beta-aminoacyl aminodiolcarbamates (U.S. Pat. No. 5,089,471); also, a variety of other peptideanalogs as disclosed in the following U.S. Pat. Nos. 5,071,837;5,064,965; 5,063,207; 5,036,054; 5,036,053; 5,034,512 and 4,894,437, andsmall molecule renin inhibitors (including diol sulfonamides andsulfinyls (U.S. Pat. No. 5,098,924), N-morpholino derivatives (U.S. Pat.No. 5,055,466), N-heterocyclic alcohols (U.S. Pat. No. 4,885,292) andpyrolimidazolones (U.S. Pat. No. 5,075,451); also, pepstatin derivatives(U.S. Pat. No. 4,980,283) and fluoro- and chloro-derivatives ofstatone-containing peptides (U.S. Pat. No. 5,066,643), enalkrein, RO42-5892, A 65317, CP 80794, ES 1005, ES 8891, SQ 34017, aliskiren(2(S),4(S),5(S),7(S)—N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)-phenyl]-octanamidhemifumarate) SPP600, SPP630 and SPP635), endothelin receptorantagonists, phosphodiesterase-5 inhibitors (e.g. sildenafil, tadalfiland vardenafil), vasodilators, calcium channel blockers (e.g.,amlodipine, nifedipine, veraparmil, diltiazem, gallopamil, niludipine,nimodipins, nicardipine), potassium channel activators (e.g.,nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam),lipid lowering agents e.g., HMG-CoA reductase inhibitors such assimvastatin and lovastatin which are marketed as ZOCOR® and MEVACOR® inlactone pro-drug form and function as inhibitors after administration,and pharmaceutically acceptable salts of dihydroxy open ring acidHMG-CoA reductase inhibitors such as atorvastatin (particularly thecalcium salt sold in LIPITOR®), rosuvastatin (particularly the calciumsalt sold in CRESTOR®), pravastatin (particularly the sodium salt soldin PRAVACHOL®), cerivastatin, and fluvastatin (particularly the sodiumsalt sold in LESCOL®); a cholesterol absorption inhibitor such asezetimibe (ZETIA®) and ezetimibe in combination with any other lipidlowering agents such as the HMG-CoA reductase inhibitors noted above andparticularly with simvastatin (VYTORIN®) or with atorvastatin calcium;HDL-raising drugs, (e.g., niacin and nicotinic acid receptor agonists,and extended- or controlled-release versions thereof, and/or with anHMG-CoA reductase inhibitor; niacin receptor agonists such as acipimoxand acifran, as well as niacin receptor partial agonists; glucagonreceptor antagonists (e.g., MK-3577, MK-0893, LY-2409021 and KT6-971);bile acid sequestering agents (e.g., colestilan, colestimide,colesevalam hydrochloride, colestipol, cholestyramine, anddialkylaminoalkyl derivatives of a cross-linked dextran), acylCoA:cholesterol acyltransferase inhibitors, (e.g., avasimibe); agentsintended for use in inflammatory conditions, such as aspirin,non-steroidal anti-inflammatory drugs or NSAIDs, glucocorticoids, andselective cyclooxygenase-2 or COX-2 inhibitors; glucokinase activators(GKAs) (e.g., AZD6370); inhibitors of 11β-hydroxysteroid dehydrogenasetype 1, (e.g., such as those disclosed in U.S. Pat. No. 6,730,690, andLY-2523199); CETP inhibitors (e.g., anacetrapib, evacetrapib, andtorcetrapib); inhibitors of fructose 1,6-bisphosphatase, (e.g., such asthose disclosed in U.S. Pat. Nos. 6,054,587; 6,110,903; 6,284,748;6,399,782; and 6,489,476); inhibitors of acetyl CoA carboxylase-1 or 2(ACC1 or ACC2); PCSK9 inhibitors; GPR-40 partial agonists; SCDmodulators; inhibitors of fatty acid synthase; amylin and amylinanalogues (e.g., pramlintide); including pharmaceutically acceptablesalt forms of the above active agents where chemically possible.

Furthermore, the present disclosure contemplates combination therapywith agents and methods for promoting weight loss, such as agents thatstimulate metabolism or decrease appetite, and modified diets and/orexercise regimens to promote weight loss.

The complexes of the present disclosure may be used in combination withone or more other agent in any manner appropriate under thecircumstances. In one embodiment, treatment with the at least one activeagent and at least one complex of the present disclosure is maintainedover a period of time. In another embodiment, treatment with the atleast one active agent is reduced or discontinued (e.g., when thesubject is stable), while treatment with a complex of the presentdisclosure is maintained at a constant dosing regimen. In a furtherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), while treatment withthe complex(es) of the present disclosure is reduced (e.g., lower dose,less frequent dosing or shorter treatment regimen). In yet anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), and treatment with thecomplex of the present disclosure is increased (e.g., higher dose, morefrequent dosing or longer treatment regimen). In yet another embodiment,treatment with the at least one active agent is maintained and treatmentwith a complex of the present disclosure is reduced or discontinued(e.g., lower dose, less frequent dosing or shorter treatment regimen).In yet another embodiment, treatment with the at least one active agentand treatment with the complex (es) of the present disclosure arereduced or discontinued (e.g., lower dose, less frequent dosing orshorter treatment regimen).

Dosing

The complexes of the present disclosure may be administered to a subjectin an amount that is dependent upon, for example, the goal of theadministration (e.g., the degree of resolution desired); the age,weight, sex, and health and physical condition of the subject to betreated; the nature of the polypeptide, and/or formulation beingadministered; the route of administration; and the nature of thedisease, disorder, condition or symptom thereof (e.g., the severity ofthe dysregulation of glucose/insulin and the stage of the disorder). Thedosing regimen may also take into consideration the existence, nature,and extent of any adverse effects associated with the agent(s) beingadministered. Effective dosage amounts and dosage regimens can readilybe determined from, for example, safety and dose-escalation trials, invivo studies (e.g., animal models), and other methods known to theskilled artisan.

In general, dosing parameters dictate that the dosage amount be lessthan an amount that could be irreversibly toxic to the subject (i.e.,the maximum tolerated dose, “MTD”) and not less than an amount requiredto produce a measurable effect on the subject. Such amounts aredetermined by, for example, the pharmacokinetic and pharmacodynamicparameters associated with absorption, distribution, metabolism, andexcretion (“ADME”), taking into consideration the route ofadministration and other factors.

An effective dose (ED) is the dose or amount of an agent that produces atherapeutic response or desired effect in some fraction of the subjectstaking it. The “median effective dose” or ED50 of an agent is the doseor amount of an agent that produces a therapeutic response or desiredeffect in 50% of the population to which it is administered. Althoughthe ED50 is commonly used as a measure of reasonable expectance of anagent's effect, it is not necessarily the dose that a clinician mightdeem appropriate taking into consideration all relevant factors. Thus,in some situations the effective amount is more than the calculatedED50, in other situations the effective amount is less than thecalculated ED50, and in still other situations the effective amount isthe same as the calculated ED50.

In addition, an effective dose of the complex(es) of the presentdisclosure may be an amount that, when administered in one or more dosesto a subject, produces a desired result relative to a healthy subject.For example, an effective dose may be one that, when administered to asubject having elevated plasma glucose and/or plasma insulin, achieves adesired reduction relative to that of a healthy subject by at leastabout 10%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or more than 80%.

An appropriate dosage level will generally be about 0.001 to 100 mg/kgof patient body weight per day, which can be administered in single ormultiple doses. In some embodiments, the dosage level will be about 0.01to about 25 mg/kg per day, and in other embodiments about 0.05 to about10 mg/kg per day. A suitable dosage level may be about 0.01 to 25 mg/kgper day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg perday. Within this range, the dosage may be 0.005 to 0.05, 0.05 to 0.5 or0.5 to 5.0 mg/kg per day.

For administration of an oral agent, the compositions can be provided inthe form of tablets, capsules and the like containing from 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0,15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0,500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the activeingredient. The complex may be administered on a regimen of, forexample, 1 to 4 times per day, and often once or twice per day.

The dosage of the complex(es) of the present disclosure may be repeatedat an appropriate frequency, which may be in the range of once per dayto once every month, depending on the pharmacokinetics of the complex(e.g. half-life) and the pharmacodynamic response (e.g. the duration ofthe therapeutic effect of the complex). In some embodiments, dosing isfrequently repeated between once per week, once every two weeks, onceevery month. In other embodiments, complex may be administeredapproximately once per month.

In certain embodiments, the dosage of the disclosed complex is containedin a “unit dosage form”. The phrase “unit dosage form” refers tophysically discrete units, each unit containing a predetermined amountof a complex of the present disclosure, either alone or in combinationwith one or more additional agents, sufficient to produce the desiredeffect. It will be appreciated that the parameters of a unit dosage formwill depend on the particular agent and the effect to be achieved.

Kits

The present disclosure also contemplates kits comprising the disclosedcomplex (es), and pharmaceutical compositions thereof. The kits aregenerally in the form of a physical structure housing variouscomponents, as described below, and may be utilized, for example, inpracticing the methods described above (e.g., administration of acomplex to a subject in need of weight reduction).

A kit can include one or more of the complex(es) disclosed herein(provided in, e.g., a sterile container), which may be in the form of apharmaceutical composition suitable for administration to a subject. Thecomplex(es) can be provided in a form that is ready for use or in a formrequiring, for example, reconstitution or dilution prior toadministration. When the complex (es) are in a form that needs to bereconstituted by a user, the kit may also include buffers,pharmaceutically acceptable excipients, and the like, packaged with orseparately from the complex (es). When combination therapy iscontemplated, the kit may contain the several agents separately or theymay already be combined in the kit. Each component of the kit can beenclosed within an individual container and all of the variouscontainers can be within a single package. A kit of the presentdisclosure can be designed for conditions necessary to properly maintainthe components housed therein (e.g., refrigeration or freezing).

A kit may contain a label or packaging insert including identifyinginformation for the components therein and instructions for their use(e.g., dosing parameters, clinical pharmacology of the activeingredient(s), including mechanism of action, pharmacokinetics andpharmacodynamics, adverse effects, contraindications, etc.). Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert may be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampoule, tube or vial). Exemplary instructions include thosefor reducing or lowering blood glucose, treatment of hyperglycemia,treatment of diabetes, etc. with the disclosed Modulators, andpharmaceutical compositions thereof

Labels or inserts can additionally include, or be incorporated into, acomputer readable medium, such as a disk (e.g., hard disk, card, memorydisk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape,or an electrical storage media such as RAM and ROM or hybrids of thesesuch as magnetic/optical storage media, FLASH media or memory-typecards. In some embodiments, the actual instructions are not present inthe kit, but means for obtaining the instructions from a remote source,e.g., via the internet, are provided.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.), but someexperimental errors and deviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: bp=base pair(s); kb=kilobase(s);pl=picoliter(s); s or sec=second(s); min=minute(s); h or hr=hour(s);aa=amino acid(s); kb=kilobase(s); nt=nucleotide(s); ng=nanogram;μg=microgram; mg=milligram; g =gram; kg=kilogram; dl or dL=deciliter; μlor μL=microliter; ml or mL=milliliter; l or L=liter; μM=micromolar;mM=millimolar; M=molar; kDa=kilodalton; i.m.=intramuscular(ly);i.p.=intraperitoneal(ly); s.c.=subcutaneous(ly); bid=twice daily;HPLC=high performance liquid chromatography; BW=body weight; U=unit;ns=not statistically significant; PG=fasting plasma glucose; FPI=fastingplasma insulin; ITT=insulin tolerance test; PTT=pyruvate tolerance test;oGTT=oral glucose tolerance test; GSIS=glucose-stimulated insulinsecretion; PBS=phosphate-buffered saline; PCR=polymerase chain reaction;NHS=N-Hydroxysuccinimide; DMEM=Dulbeco's Modification of Eagle's Medium;GC=genome copy; EDTA=ethylenediaminetetraacetic acid.

Materials and Methods

The following methods and materials were used in the Examples below:

Animals. Diet-induced obese (DIO) male C57BL/6J mice (The JacksonLaboratory, Bar Harbor, Me.) were maintained on a high-fat diet (D12492,Research Diets, Inc, New Brunswick, N.J.) containing 60 kcal % fat, 20kcal % protein and 20 kcal % carbohydrate for 12-20 weeks. All animalstudies were approved by the NGM Institutional Animal Care and UseCommittee. DIO C57BL/6J mice offer a human-like model of obesity, wherethe obesity is based upon excessive intake of calories. C57BL/6J miceare obesity-prone in which pronounced weight gain, as well ashyperinsulinemia and sometimes hyperglycemia, is observed. The strain ismost-commonly used mouse strain for modeling diet-induced obesity.(Nilsson C., et al., Acta Pharmacologica Sinica (2012) 33: 173-181).

Nucleic Acid and Amino Acid Sequences. GenBank Accession No. BC000529.2sets forth the cDNA of ORF encoding human GDF15 variants, and GenBankAccession No. NP_004855.2 sets forth the amino acid sequence encoded bythe cDNA. The cDNA for the Fc-fusion partner was purchased at InvivoGen(pFUSE-CHIg-hG1, GenBank: AY623427.1, protein ID=AAT49050) and modifiedas indicated. The amino acid sequence of the Fc-fusion partner encodedby the pFUSE-CHIg-hG1 vector is:

(SEQ ID NO: 55) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Construction of Expression Constructs. The mammalian expression vectorpTT5 (National Research Council Canada) was modified by inserting aKozak element and human IgK-Signal Peptide sequence:(CACCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGT)(SEQ ID NO: 56) between the PmeI and EcoRI site. While both restrictionsites were eliminated an AgeI site was created for further in-framecloning of secreted factors. For single fragment insertion (e.g., Fcportion of human IgG1), In-Fusion technology (Clontech) was used. Forthe insertion of two or more PCR generated fragments (i.e.hIgG1-Fc+GDF15) we used Gibson Assembly Master Mix (NEB) according tomanufactures protocols. All PCR fragments were amplified by Sapphire PCRmix and gel-purified using Qiagen Gel Extraction kit. TOP10Electro-competent cells (Life Technologies) were transformed withcloning reactions, plated on LB-agar plates containing carbenicillin andincubated over night at 37° C. Single colonies were picked and analyzedby sequencing. DNA from positive colonies was amplified (DNA-Maxi-prep,Qiagen), fully sequence confirmed and used to transfect mammalian cellsfor recombinant protein expression.

To create specific muteins, site directed mutagenesis was performed witheither QuikChange Lightning or QuikChange Lightning Multi Site-DirectedMutagenesis Kits (Agilent) and appropriate primers, followingmanufactures protocols.

(Fc/Fc)-GDF15 Fusion Molecule, Wild Type GDF15, and GDF15-glycomuteinExpression. All molecules were recovered from transiently transfected inExpi 293F cells (Invitrogen Corporation, Carlsbad, Calif.). Cells wereroutinely subcultured in Expi expression medium (Invitrogen) andmaintained as suspension cultures in shake flasks of varying sizes.Typically, cells were subcultured at a cell density of 5e5 viablecells/ml and grown for 3 days before subculturing. The flasks weremaintained in a humidified CO₂ incubator (37° C. and 5% CO₂) on NewBrunswick shaker platforms (New Brunswick Scientific Company, Edison,N.J.) at an agitation rate of 110 RPM.

Transfections were performed when the cell density of the culturereached 2.5e6 viable cells/mL at greater than 95% viability. Typically,for 50 mL transfection, 2.5e6 cells/mL×50 mL cells were inoculated in a250 mL shaker flask in 42.5 mL culture volume. Fifty micrograms (50 μm)plasmid DNA consisting of the expression vector containing the gene ofinterest was first diluted in 2.5 mL OPTI-MEM reduced-serum medium(Invitrogen). Simultaneously, Expifectamine transfection reagent(Invitrogen), 2.67 times the volume (of the amount of plasmid DNA) wasalso diluted in 2.5 mL OPTI-MEM reduced-serum medium. After a 5 minincubation at room temperature, the diluted transfection reagent wasslowly added to the diluted plasmid DNA to form transfection competentcomplexes. After a further 20 min incubation period at room temperature,5 mL of the transfection complex was added to the 42.5 mL cell culture.The transfected cells were then placed in the humidified CO₂ incubatoron an orbital shaker maintained at 110 RPM. Twenty-four hourspost-transfection, the transfected culture was fed with 250 μL enhancer1 solution (Invitrogen) and 2.5 mL enhancer 2 solution (Invitrogen). Theculture was then replaced in the humidified CO₂ incubator on an orbitalshaker. Six-to-seven days post-transfection, cultures were harvested bycentrifugation at 3000 RPM for 30 min before being filtered through a0.2 μm filter (Nalgene). Samples were then analyzed on a commassie staingel for expression.

Purification of Recombinant Protein. (Fc/Fc)-GDF15 molecules expressedinto conditioned media (CM) were assessed for recovery and activityfollowing purification. CM was passed over mAb SelectSuRe column (GE) ata loading capacity of no greater than 20 mg/mL of resin. CM volumesranged from 50 mL-1000 mL for assessment of recoveries. Following mAbSelectSuRe loading of CM, the column was washed with 5-10 column volumesof 1×PBS (Corning Cellgro) followed by step elution with low pH Glycinebuffer (Polysciences Inc). Following elution, the (Fc/Fc)-GDF15 poolswere pH neutralized with 1M Tris pH 8.0 (Teknova) and then injected ontoa Superdex200 (GE) column pre-equilibrated in 1×PBS (Corning Cellgro).Fractions of (Fc/Fc)-GDF15 intact, fully assembled molecules were pooledand assessed for purity and quantitated via A280 methods usingappropriate extinction coefficient and molecular weights to determinerecovery based on starting CM volumes. The fully assembled moleculeswere dimer-dimer complex of two heterodimers. Each heterodimer having aFc associated with Fc-GDF15 glycomutein via knob in hole interaction,and two heterodimers associated via GDF15-GDF15 interaction.

Purification of WT GDF15 and GDF15 Glycomuteins. Wild type GDF15 and theGDF15 glycomuteins not conjugated to Fc were purified from culturedmedia using ion-exchange capture. WT GDF15 and GDF15 glycomuteins wereeluted using a gradient of appropriate salt/pH conducive for optimalelution and separation from host cell protein impurities. All GDF15molecules were then further purified using GE HiTrap Phenyl HP at pH 8.0using a decreasing linear gradient of ammonium sulfate. Fractions wereassessed and pooled based on purity and glycosylation properties viagel-shift on non-reduced SDS-PAGE gels. Similar to the (Fc/Fc)-GDF15molecules, the wild type GDF15 and the GDF15 glycomuteins were expressedusing the IgK signal peptide.

Example 1: Design of Heterodimeric Knob-in-hole (Fc/Fc)-GDF15 FusionMolecules

Fc-GDF15 designs are described in FIG. 1 and primary sequences aredepicted below (Constructs B1a/b-B19a/b). To achieve productive assemblyof Fc-GDF15 molecules, an efficient system was designed to allow forFc/Fc dimerization whilst allowing for GDF15/GDF15 dimerization. Toavoid mis-folding and aggregation potential of single-chain Fc-GDF15, aheterodimeric fusion partner was designed for the Fc/Fc interaction toallow for high fidelity GDF15/GDF15 homodimerization. Knob-in-hole Fc/Fcheterodimers were designed to address GDF15 assembly and secretion fromExpi 293F transient systems. Fc/Fc heterodimeric knob-in-hole systemswere assessed using a [T366Y (knob)//Y407T (hole)] or a [T366W(knob)//T3665-L368A-Y407V (hole)] system, coupled with a (G₄S)_(n)linker (n=2, 3, 4, or 5) and GDF15. It is noted that the numbering ofthe amino acid position in the CH3 domain of Fc is based on the EUnumbering system (Edelman, G. M. et al., Proc. Natl. Acad. USA, 63,78-85 (1969)). In all cases, the Fc-fusion (knob/hole) partner wascoupled to the N-terminus of mature GDF15 comprising amino acid residuesA1-I112, R2-I112, N3-I112, G4-I112, D5-I112, H6-I112, or C7-I112.Truncations of the N-terminus of GDF15 (Δ1=R2-I112, Δ2=N3-I112,Δ3=G4-I112, Δ4=D54112, Δ5=H6-I112, or Δ6=C7-I112) were incorporated forstability enhancement as the sequence at the N-terminus (ARNGDH, SEQ IDNO: 95) has previously been demonstrated as a site of proteolyticsusceptibility and N-terminal truncations provide superior stability vs.GDF15 that does not include these N-terminal truncations.

FIGS. 1A-1D describe the placement of the knob vs. hole on the Fc-GDF15(chain A), coupled with the corresponding hole vs. knob on theheterodimeric Fc partner (chain B), coupled with either a wild-type IgGhinge containing two intermolecular disulfide bonds or without the hingedomain (Δhinge). For the Fc heterodimeric knob or hole A/B chains, an AAmutation (APELLGGP (SEQ ID NO: 96)

APALAGGP (SEQ ID NO: 97)) was introduced for removal of IgG1 effectorfunctionality. (Fc/Fc)-GDF15 heterodimeric knob-in-hole designs wereexpression profiled for assembly and are reported in FIG. 2A. In allcases, transient expression of knob-in-hole (Fc/Fc)-GDF15 resulted inrecoveries, following purification, between 0 mg/L and 74.9 mg/L ofcorrectly assembled product (0=aggregates/no expression, <25 mg/L, 25mg/L-49.9 mg/L, 50 mg/L-74.9 mg/L, 75 mg/L-99.0 mg/L, >100 mg/L). In allcases, assembly and secretion of the knob-in-hole heterodimericFc/Fc-GDF15 molecules were accompanied with various contaminating levelsof mis-folded homodimeric species such as Fc(hole):Fc(hole),Fc(knob):Fc(knob), Fc(knob)-GDF15:Fc(knob)-GDF15 andFc(hole)-GDF15:Fc(hole)-GDF15. Based on expression profiling, the T366W(knob) placed on the Fc-GDF15 chain, coupled with the T366S-L368A-Y407V(hole) on the heterodimeric Fc partner chain (FIG. 1D) was found toproduce a product with maximal stability and minimized mis-pairing ofFc/Fc-homodimeric products (FIG. 2A—variant B5a/B5b). This design wasthe focus of further expression engineering and optimization.

Variant B5a/B5b recovery from transiently expressed Expi 293F sourceprovided recoveries in the range of 0.0 mg/L to 24.9 mg/L. To enhanceexpression, assembly & recoveries, N-glycosylation sites were introducedwithin the mature sequence of GDF15 (FIG. 1F). In the designedconstructs, the presence of a single N-linked glycan consensus site onGDF15 significantly improved expression, assembly and recovery of thefully mature (Fc/Fc)-GDF15 knob-in-hole heterodimer B5a/B5b (FIG.2A—variants B9a/B9b to B19a/B19b). The linker length was found to beoptimal when n=5 for (G₄S)_(n) for receptor binding & activity via an invitro assay. The presence of a glycan on position DST completely removesa primary deamidation site on position N3 of mature GDF15 and appears tofurther enhance stability of the molecule as is evidenced in Example 2.

The presence of N-linked glycans within the sequence of GDF15 isproposed to help expression and minimize mis-folded products fromaccumulating due to increased residence time in the EndoplasmicReticulum and Golgi Apparatus during the secretory process. Thisadditional residence time is proposed to have a beneficial effect onfolding kinetics and allows for significantly improved hetero-dimeric(Fc/Fc) knob-in-hole pairing and recoveries from mammalian tissueculture.

The sequences of the variants (B1a/b-B19a/b) are provided below. In thesequences depicted below, human IgK signal peptide is in lower casefollowed by Fc sequence. In the sequences that also include linker andGDF15 sequence, the Fc sequence is followed by linker sequence(underlined) which is followed by GDF15 sequence (in bold). Thenumbering of the position of amino acid substitutions in the Fc sequenceis based on EU numbering, the substitutions with reference to the aminoacid present at corresponding position in human IgG1Fc (SEQ ID NO: 2).The numbering of N-terminal deletion in GDF15 sequence and amino acidsubstitution(s) is with reference to wild type human mature GDF15 (SEQID NO: 1).

B1a: hIgK-hIgG1-Fc(AA)(T366Y)-(G₄S)₅-ΔN3-GDF15 (G4-I112) (SEQ ID NO: 57)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI B1b: hI2K-hIgG1-Fc(AA)(Y407T)(SEQ ID NO: 58)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B2a: hI2K-hIgG1-Fc(AA)(Y407T)-(G₄S)₅-ΔN3-GDF15 (G4-I112)(SEQ ID NO: 59)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI B2b: hIgK-hIgG1-Fc(AA)(T366Y)(SEQ ID NO: 60)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B3a: hIgK-hIgG1-Fc(Δhinge, AA)(T366Y)-(G₄S)₅-ΔN3-GDF15(G4-I112) (SEQ ID NO: 61)mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI B3b: hIgK-hIgG1-Fc(Δhinge, AA)(Y407T)(SEQ ID NO: 62)mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKB4a: hIgK-hIgG1-Fc(Δhinge, AA)(Y407T)-(G₄S)₅-ΔN3-GDF15 (G4-I112)(SEQ ID NO: 63)mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI B4b: hIgK-hIgG1-Fc(Δhinge, AA)(T366Y)(SEQ ID NO: 64)mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKB5a: hI2K- hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15 (G4-I112)(SEQ ID NO: 65)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB5b: hI2K-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 66)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKB6a: hI2K-hIgG1-Fc(AA) (T366S)(L368A)(Y407V)-(G₄S)₅-ΔN6-GDF15 (C7-I112)(SEQ ID NO: 67)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS CPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI B6b: hI2K-hIgG1-Fc(AA)(T366W)(SEQ ID NO: 68)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B7a: hIgK-hIgG1-Fc(Δhinge, AA)(T366W)-(G₄S)₅-ΔN3-GDF15(G4-I112) (SEQ ID NO: 69)mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB7b: hIgK-hIgG1-Fc(Δhinge AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 70)mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKB8a: hIgK-hIgG1-Fc(Δh, AA) (T366S)(L368A)(Y407V)-(G₄S)₅-ΔN6-GDF15 (C74-I12) (SEQ ID NO: 71)mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGG SCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI B8b: hIgK-hIgG1-Fc(Δh, AA)(T366W)(SEQ ID NO: 72)mdmrvpaqllgllllwlrgarcAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKB9a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₃-GDF15 (A1-I112) (D5T)(SEQ ID NO: 73)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS ARNGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB9b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 74)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B10a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₄-GDF15 (A1-I112)(D5T) (SEQ ID NO: 75)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGS ARNGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB10b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 76)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B11a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₅-GDF15 (A1-I112)(D5T) (SEQ ID NO: 77)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS ARNGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB11b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 76)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B12a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₂-ΔN2-GDF15 (N3I-112)(D5T) (SEQ ID NO: 78)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGS NGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI B12b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V)(SEQ ID NO: 79)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B13a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN2-GDF15 (N3-I112)(D5T) (SEQ ID NO: 80)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS NGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB13b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 81)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B14a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15 (G4-I112)(R21N) (SEQ ID NO: 82)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVNASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB14b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 83)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B15a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15 (G4-112)(S23N/E25T) (SEQ ID NO: 84)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRANLTDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB15b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 85)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B16a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15 (G4-I112)(F52N/A54T) (SEQ ID NO: 86)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQNRTANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB16b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 87)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B17a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15 (G4-I112)(R53N/A55T) (SEQ ID NO: 88)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFNATNMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIB17b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 89)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B18a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15 (G4-I112)(K91N/D93T) (SEQ ID NO: 90)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQNTTTGVSLQTYDDLLAKDCHCIB18b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 91)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B19a: hIgK-hIgG1-Fc(AA)(T366W)-(G₄S)₅-ΔN3-GDF15 (G4-I112)(D93N/G95T) (SEQ ID NO: 92)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTNTTVSLQTYDDLLAKDCHCIB19b: hIgK-hIgG1-Fc(AA)(T366S)(L368A)(Y407V) (SEQ ID NO: 93)mdmrvpaqllgllllwlrgarcDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Sequences of the wild type human mature GDF15 (SEQ ID NO: 1) and GDF15glycomuteins listed in FIG. 2B are as follows:

IgK-Wild type human mature GDF15 (SEQ ID NO: 108)mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI IgK-GDF15-glycomutein R21N(SEQ ID NO: 109) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVNASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI IgK-GDF15-glycomutein R53N/A55T(SEQ ID NO: 110) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFNATNMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI IgK-GDF15-glycomutein S64N/H66T(SEQ ID NO: 111) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTNLTRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI IgK-GDF15-glycomutein P70N(SEQ ID NO: 112) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKNDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI IgK-GDF15-glycomutein Q90N(SEQ ID NO: 113) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLINKTDTGVSLQTYDDLLAKDCHCI IgK-GDF15-glycomutein K91N/D93T(SEQ ID NO: 114) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQNTTTGVSLQTYDDLLAKDCHCI IgK-GDF15-glycomutein D93N/G95T(SEQ ID NO: 115) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTNTTVSLQTYDDLLAKDCHCI IgK-GDF15-glycomutein G95N(SEQ ID NO: 116) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTNVSLQTYDDLLAKDCHCI IgK-GDF15-glycomutein S97N/Q99T(SEQ ID NO: 117) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVNLTTYDDLLAKDCHCI IgK-GDF15-glycomutein L98N(SEQ ID NO: 118) mdmrvpaqllgllllwlrgarcARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSNQTYDDLLAKDCHCI

The GDF15 molecules were expressed using the IgK signal peptide, whichis cleaved off from the secreted polypeptide by a signal peptidaseexpressed by the 293 cells. The recovery of the wild type human matureGDF15 (SEQ ID NO: 1) and GDF15 glycomuteins is listed in FIG. 2B.

Exemplary GDF15 glycomuteins that may be expressed as Fc-Fc(knob/hole)GDF15 glycomuteins are described in U.S. Ser. No. 14/811,578 filed onJul. 28, 2015.

Example 2: Effects of (Fc/Fc)-GDF15 Fusion Molecules on Body Weight andFood Intake in DIO Mouse Model

The effects of a subcutaneously administered fusion molecule havingrecombinant Fc-heterodimer fused to recombinant human GDF15 (i.e., acomplex of two heterodimers, each heterodimer having a Fc polypeptidedimerized with a Fc-GDF15 glycomutein polypeptide) on body weight wereevaluated over a 35 day period. Briefly, the fusion molecules B9a/B9b,B11a/B11b and B13a/B13b were administered weekly for 21 days at doses of0.4 nmol/kg and 4 nmol/kg as a single subcutaneous bolus injection (10mL/kg) to DIO mice weighing approximately 35-40 g. Followingadministration of vehicle control or the fusion molecules, body weightreduction was monitored at various time points over a 35 day time periodwhich comprised 21 days of protein dosing followed by a 14 day wash out(post-dose) to monitor efficacy.

As depicted in FIGS. 3-6, administration of the Fc fusion molecules(heterodimer-heterodimer complex) at a dose of 0.4 nmol/kg and 4 nmol/kgresulted in significant body weight reduction. In each group of mice,n=6 and p-values (*, p<0.05; **, p<0.01; ***, p<0.001, ns=notsignificant) were determined by student's unpaired T-test comparing tovehicle control group at each specified time point. As depicted in FIG.7, total body weight with SEM analysis is shown at each time pointsampling for all groups. As depicted in FIG. 8, changes in body weight(g) with SEM analysis and p-values are shown at each time point samplingfor all groups. As depicted in FIG. 9, percent changes in body weight(%) with SEM analysis and p-values are shown at each time point samplingfor all groups.

As depicted in FIGS. 3 & 5, there is an observed increased efficacy inbody weight reduction for B13a/B13b as compared to B9a/B9b and B11a/B11bin the 0.4 nmol/kg dose study. The increased in vivo efficacy forB13a/B13b is attributed to enhanced stability due to the truncation ofthe 2 N-terminal residues of GDF15 (ΔAR).

Particular embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Upon reading the foregoing, description, variations of the disclosedembodiments may become apparent to individuals working in the art, andit is expected that those skilled artisans may employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A first nucleic acid and a second nucleic acid,the first nucleic acid encoding a first polypeptide comprising from theN-terminus to the C-terminus: a human IgG1 Fc comprising a hinge regionand a CH3 sequence comprising a substitution selected from the groupconsisting of Q347W/Y, T366W/Y, and T394W/Y, according to EU numbering,a linker, and an N-glycosylated GDF15 mutein comprising the amino acidsequence of SEQ ID NO: 129; and the second nucleic acid encoding asecond polypeptide comprising a human IgG1 Fc comprising a hinge regionand a CH3 sequence comprising a substitution selected from the groupconsisting of T366S, L368A, T394S, F405T/V/A, and Y407T/V/A, accordingto EU numbering, wherein the first and second polypeptides each form afirst heterodimer and a second heterodimer, wherein the first and secondheterodimers form a complex.
 2. The first and second nucleic acids ofclaim 1, wherein each of the nucleic acids is operably linked to anexpression control element that confers expression of the first andsecond polypeptides from the first and second nucleic acids,respectively.
 3. The first and second nucleic acids of claim 1, whereinthe linker comprises the sequence Glycine-Glycine-Glycine-Glycine-Ser(G₄S)_(n), and wherein n=2-10.
 4. The first and second nucleic acids ofclaim 1, wherein the linker comprises the sequence (G₄S)₃.
 5. The firstand second nucleic acids of claim 1, wherein the linker comprises thesequence (G₄S)₅.
 6. The first and second nucleic acids of claim 1,wherein the first polypeptide comprises the substitution T366W/Y and thesecond polypeptide comprises the substitution Y407T/V/A.
 7. The firstand second nucleic acids of claim 1, wherein the N-glycosylated GDF15mutein comprises the amino acid sequence of SEQ ID NO:
 128. 8. The firstand second nucleic acids of claim 1, wherein the first polypeptidecomprises the amino acid sequence set forth in SEQ ID NO:
 3. 9. Thefirst and second nucleic acids of claim 8, wherein the secondpolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4.10. The first and second nucleic acids of claim 1, wherein the firstpolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.11. The first and second nucleic acids of claim 10, wherein secondpolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6.12. The first and second nucleic acids claim 1, wherein the firstpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:80.
 13. The first and second nucleic acids of claim 12, wherein thesecond polypeptide comprises the amino acid sequence set forth in SEQ IDNO:
 81. 14. A first vector and a second vector, the first vectorcomprising the first nucleic of claim 1 and the second vector comprisingthe second nucleic acid of claim
 1. 15. The first and second vectors ofclaim 14, wherein each of the vectors comprises a viral vector.
 16. Ahost cell comprising the first and second nucleic acids of claim
 1. 17.A host cell comprising the first and second vectors of claim
 14. 18. Ahost cell expressing a complex comprising: a first heterodimer and asecond heterodimer, each of the first heterodimer and second heterodimercomprising: (i) a first polypeptide comprising from the N-terminus tothe C-terminus: a human IgG1 Fc comprising a hinge region and a CH3sequence comprising a substitution selected from the group consisting ofQ347W/Y, T366W/Y, and T394W/Y, according to EU numbering, a linker, andan N-glycosylated GDF15 mutein comprising the amino acid sequence of SEQID NO: 129, and (ii) a second polypeptide comprising a human IgG1 Fccomprising a hinge region and a CH3 sequence comprising a substitutionselected from the group consisting of T366S, L368A, T394S, F405T/V/A,and Y407T/V/A, according to EU numbering.
 19. A host cell expressing acomplex comprising: a first heterodimer and a second heterodimer, eachof the first heterodimer and second heterodimer comprising: a firstpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:5; and a second polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 6, wherein the first polypeptide is N-glycosylated, andwherein the first polypeptide is dimerized with the second polypeptidevia one or two disulphide bonds formed between hinge regions present inIg Fc sequences in the first and second polypeptides.
 20. A method forisolating a complex, the method comprising: culturing the host cell ofclaim 16 for expression of the first and second polypeptides; andisolating a complex comprising the first polypeptide and the secondpolypeptide.
 21. A method for isolating a complex, the methodcomprising: culturing the host cell of claim 17 for expression of thefirst and second polypeptides; and isolating a complex comprising thefirst polypeptide and the second polypeptide.
 22. A nucleic acidencoding: (i) a first polypeptide comprising from the N-terminus to theC-terminus: a human IgG1 Fc comprising a hinge region and a CH3 sequencecomprising a substitution selected from the group consisting of Q347W/Y,T366W/Y, and T394W/Y, according to EU numbering, a linker, and anN-glycosylated GDF15 mutein comprising the amino acid sequence of SEQ IDNO: 129, and (ii) a second polypeptide comprising a human IgG1 Fccomprising a hinge region and a CH3 sequence comprising a substitutionselected from the group consisting of T366S, L368A, T394S, F405T/V/A,and Y407T/V/A, according to EU numbering, wherein the first and secondpolypeptides are present in each of a first heterodimer and a secondheterodimer, wherein the first and second heterodimers form a complex.23. The nucleic acid of claim 22, wherein the nucleic acid is operablylinked to an expression control element that confers expression of thefirst and second polypeptides from the nucleic acid.
 24. A vectorcomprising the nucleic acid of claim
 22. 25. The vector of claim 24,wherein the vector comprises a viral vector.
 26. A host cell comprisingthe nucleic acid of claim
 22. 27. A host cell comprising the vector ofclaim
 24. 28. A method for isolating a complex, the method comprising:culturing the host cell of claim 26; and isolating a complex comprisingthe first polypeptide and the second polypeptide.
 29. A method forisolating a complex, the method comprising: culturing the host cell ofclaim 27; and isolating a complex comprising the first polypeptide andthe second polypeptide.
 30. The first and second nucleic acids of claim1, wherein the first polypeptide comprises the substitution T366W. 31.The first and second nucleic acids of claim 1, wherein the secondpolypeptide comprises the substitutions T366S, L368A, and Y407V.
 32. Thefirst and second nucleic acids of claim 1, wherein the first polypeptidecomprises the substitution T366W and the second polypeptide comprisesthe substitutions T366S, L368A, and Y407V.
 33. The first and secondnucleic acids of claim 32, wherein the linker comprises the sequence(G₄S)₅.
 34. The nucleic acid of claim 22, wherein the first polypeptidecomprises the substitution T366W.
 35. The nucleic acid of claim 22,wherein the second polypeptide comprises the substitutions T366S, L368A,and Y407V.
 36. The nucleic acid of claim 22, wherein the firstpolypeptide comprises the substitution T366W and the second polypeptidecomprises the substitutions T366S, L368A, and Y407V.
 37. The nucleicacid of claim 22, wherein the linker comprises the sequence (G4S)₅. 38.The nucleic acid of claim 22, wherein the first polypeptide comprisesthe amino acid sequence set forth in SEQ ID NO:
 5. 39. The nucleic acidof claim 38, wherein the second polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 4.